Several neurotoxins have been used in animal models to mimic Parkinson?s disease (PD) e.g. 6-hydroxydopamine (specific toxin for catecholaminergic neurones), methamphetamine (relative specific for dopaminergic neurones in substantia nigra), 1,methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP, toxic in mice and primates) and its neurotoxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) , tetrahydroisoquinolines, ß-carbolines, paraquate, rotenone and sodium malonate. Most of the neurotoxins inhibit complex 1 of the respiratory chain in the mitochondria leading to the production of superoxide free radical, hydrogen peroxide and hydroxyl radicals (group name: reactive oxygen species, ROS, see scheme pathogenesis of Parkinson?s disease).
We found the methamphetamine model disadvantagous because of strong variations in interindividual sensitivity of rats with some letalities and differences in the decrease of dopamine levels after application of a standard dose.
The results with sodium malonate were much less variable. Malonate is injected stereotaxically into the striatum and alternatively into medial forebrain bundle. It inhibits the enzyme succinatedehydrogenase (SDH). This mitochondrial enzyme plays a central role for energy supply of the neurone. It is part of the tricarbone cycle and the oxidative phosphorylation. Injection of soduim malonate causes lesions of dopaminergic and glutamatergic nerve terminals, and a strong decrease of ATP in the striatum. By applying an appropriate design, L-DOPA diminishes the depletion of dopamine. Substances of interest can be compared with the action of L-DOPA.
Alternatively, sodium malonate (3µmol) is injected in the medial forbrain bundle of one side. By applying amphetamine or apomorphine after 2 weeks, the lesion can by assessed in vivo before the experiment proper starts (turning of the rat to one site). The unlesioned side may serve as control.
The BC was injected by stereotaxic surgery into both substantiae nigrae of rats. We found a clear dose-dependnet decrease of the levels of dopamine, its metabolites 3,4-dihydroxyphenylalanine (DOPAC), homovanillic acid (HVA), and 3-methoxytyramine (3-MT) in the striatum.The SD was ~10%. Noradrenaline levels did not change.
Various doses of L-DOPA have been injected i.p. in rats following pretreatment with benserazide (10mg/kg i.p.). The concentrations of dopamine,DOPAC, HVA, 3-MT, 5-HT, 5-HIAA, and NA were measured in tissue samples of striatum and substantia nigra by HPLC. Optional dissections of other brain areas are possible. This method serves to compare L-DOPA, the gold standard of PD treatment, with substances of interest with respect to the activation of dopaminergic neurones.
The method is performed in the nucleus accumbens of rats. This is a brain region with dense dopaminergic nerve terminals. A microdialysis probe is inserted into the brain and perfused with artificial cerebrospinal fluid (CSF). After collection of 5 samples (basal values) the test substance is applied either orally, intraperitoneally, or directly in the brain in the freely moving rat. The dialysate is analysed by HPLC.
This is the method of choice for investigations of the time course and dose-response of changes of neurotransmitters by a test drug in specific brain regions in the awake rat.
Embryos from pregnant C57/B16 mice, gestation day (GD) 14, were used in the experiments. The mesencephalon was dissected off. The cells were cultivated in DMEM in plates or, for fluorescence microscopy, in chamber slides precoated with poly-D-lysine. On the day 8 in culture, the medium was supplemented with test compounds. This model is suited to test neurotoxic and neuroprotective properties of compounds specifically what dopaminergic neurones are concerned (e.g. survival of primary cell cultures of mesencephalic neurones, assessment of cell viability by the inner-salt tetrazolium conversion to formazan (MTT-method), LDH release (cytotoxcicity), ATP levels, necrosis/apoptosis, caspase 3 activity, mitochondrial membrane potential, formation of oxygen radicals (ROS), uptake of radiolabelled dopamine, staining of tyrosine hydroxylase by immune cytochemistry methods, a marker of dopaminergic neurones).
This model is a step closer to the ?real live? compare to the use of permanent cell lines elaborated below (cooperation with Dr. Gabriele Gille, TU Dresden).
Several cell lines have been used, e.g. neuroblastoma 2A, native and stable transfected with murine high affinity dopamine transporter (DAT), HEK 293 cells, native, stable transfected with human DAT, stable transfected with human organic cationic transporter type 1, 2, or 3, human neuroblastoma SH-SY5Y, hepatoma Hep G2 cells, and NIH 3T3 fibroblastoma cells.
The selected cells were exposed to the test compound for a certain time period. Then, they were loaded with 2?,7?-dichlorodihydrofluoresceine diacetate (H2DCFDA). The indicator substance diffuses into cells, where the acetate moieties are cleaved by intracellular esterases to form H2DCF and thereby traps it within the cells. The ROS-oxidized DCF, an indicator of intracellular ROS levels, is quantified fluorometrically.
The mitochondrial inner membrane integrity was assessed by testing the electrochemical proton gradient (ΔΨ) of the inner mitochondrial membrane. This is accomplished by measuring the uptake of the fluorescent carbocyanine dye JC-1 into mitochondria. Both increased membrane potential and lowered potential e.g. by neurotoxic substances are monitored.
The caspase-3/7 substrate rhodamine 110, exists in a profluorescent form. Upon sequential cleavage and removal of the DEDV peptides by caspase 3/7 activity the rhodamine leaving group becomes intensely fluorescent. The amount of fluorescent product generated is proportional to the amount of caspase 3/7 cleavage activity present in the sample.
DNA was extractet from the cells and analysed by agarose gel electrophoresis. DNA fragmentation by apoptotic processes are visualized by the characteristic ?ladder?.
The specificity of some neurotoxins for dopaminergic neurones arises from the DAT-mediated transport from the outside of the dopaminergic neurone into the inside. Whether the substance of interest is transported by the DAT-protein can be examined by comparing the neurotoxic potency in native and stable DAT-transfected cells. An alternative transporter protein is the organic cationic transporter (OCT), former designation: uptake 2. Meanwhile 3 different OCTs have been described with differing pharmacology. We have all 5 cell models available and used in several studies. Furthermore, the transport of some substances can be determined directly by monitoring the autofluorescence. For example, we have demonstrated a direct transport of ß-carbolines.
The method of real time RT-PCR with specific FRET-probes was established to investigate changes in mRNA levels of several members/ interplayer of the MAP cascade (RKIP, Raf, Mek, Erk) by different BCs in a human dopaminergic cell model (SH-SY5Y cells transdifferentiated with retinoic acid). The housekeeping gene hPBGD is used as standard. Additionally, we established real time RT-PCR with specific FRET probes for some dopaminergic features like DAT and both splice variants of DRD2.
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