Characterization of a Parkinson's disease rat model using an upgraded paraquat exposure paradigm.

Animal models of human diseases are crucial experimental tools to investigate the mechanisms involved in disease pathogenesis and to develop new therapies. In spite of the numerous animal models currently available that reproduce several neuropathological features of Parkinson disease (PD), it is challenging to have one that consistently recapitulates human PD conditions in both motor behaviors and biochemical pathological outcomes. Given that, we have implemented a new paradigm to expose rats to a chronic low dose of paraquat (PQ), using osmotic minipumps and characterized the developed pathologic features over time. The PQ exposure paradigm used lead to a rodent model of PD depicting progressive nigrostriatal dopaminergic neurodegeneration, characterized by a 41% significant loss of dopaminergic neuron in the substantia nigra pars compacta (SNpc), a significant decrease of 18% and 40% of dopamine levels in striatum at week 5 and 8, respectively, and a significant 1.5-fold decrease in motor performance. We observed a significant increase of microglia activation state, sustained levels of α-synucleinopathy and increased oxidative stress markers in the SNpc. In summary, this is an explorative study that allowed to characterize an improved PQ-based rat model that recapitulates cardinal features of PD and may represent an attractive tool to investigate several mechanisms underlying the various aspects of PD pathogenesis as well as for the validation of the efficacy of new therapeutic approaches that targets different mechanisms involved in PD neurodegeneration.

An easy-to-use liquid chromatography method with fluorescence detection for the simultaneous determination of five neuroactive amino acids in different regions of rat brain.

INTRODUCTION: To comprehend the normal function and pathological characteristics of certain neurological disorders it is important to evaluate the neuroactive amino acids levels in animal models. METHODS: This work describes a simple liquid chromatography-fluorescence detection (LC-FLD) method for the simultaneous determination of aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), taurine (Tau) and γ-aminobutyric acid (GABA), using methyl-L-arginine as internal standard, in samples of rat brain tissue. The five target analytes (Asp, Glu, Gln, Tau and GABA) were determined in a single chromatographic run of less 11 min after a derivatization step with o-phthalaldehyde. The derivatives were separated on a reversed-phase C18 column and detected by fluorescence at excitation and emission wavelengths of 340 and 448 nm, respectively. RESULTS: The method was validated in accordance with international guidelines on bioanalytical methods validation and it presented limits of quantification in the range of 25-50 ng mL-1 and calibration curves with determination coefficients (r2) equal to or higher than 0.9920. In addition, the precision (coefficient variation, %) and accuracy (bias, %) of the method meet the established criteria, and the stability of the analytes at the sample handling and storage conditions was demonstrated. DISCUSSION: Unlike other similar bioanalytical assays, the current method was validated using diluted biological matrix, which is advantageous in order to ensure the derivatization process integrity. Moreover, this LC-FLD method was successfully applied for the determination of the compounds of interest in different rat brain tissue regions (frontal cortex, amygdala, hippocampus, cerebellum and striatum). Thus, this bioanalytical assay represents a useful tool to support multiple nonclinical studies in the field of neurosciences, requiring the quantitative profiling and pattern analysis of neuroactive amino acids.

Determination of catecholamines and endogenous related compounds in rat brain tissue exploring their native fluorescence and liquid chromatography.

The profiling analysis of catecholamines and their metabolites in brain tissue offers a crucial key to understand their functions in the body and the opportunity to follow up neural diseases. A rapid and simple liquid chromatography-fluorescence detection (LC-FLD) method was developed and validated for simultaneously measuring several catecholamines and endogenous related compounds in the rat brain tissue samples. The target analytes measured in this bioanalytical assay were levodopa (L-DOPA), dopamine (DA), norepinephrine (NE), epinephrine (E), 3-O-methyldopa (3-O-MD), and homovanillic acid (HVA), being the 3,4-dihydroxybenzylamine (DHBA) used as internal standard (IS). The six analytes (L-DOPA, DA, NE, E, 3-O-MD and HVA) can be determined in a single chromatographic run of less than 12min, and all the compounds (analytes and IS) were detected using their native fluorescence and monitored at excitation/emission wavelengths of 279nm/320nm, respectively. The chromatographic and detection conditions were experimentally optimized and then several validation parameters (linearity, limits of quantification and detection, precision and accuracy, recovery, stability and selectivity) were examined. In accordance with the international guidelines of the Food and Drug Administration and European Medicines Agency the method described herein exhibited limits of quantification in the range of 2-25ngmL-1, linearity in wide concentration ranges (r2≥0.994), and acceptable precision (coefficient variation ≤8.76%) and accuracy (bias ±14.65%) levels. Since the bioanalytical procedure does not involve pre-purification or derivatization of the sample, the absolute recovery was found to be around 100%. Moreover, the developed LC-FLD method was successfully applied for the determination of the compounds of interest in tissue samples of different rat brain regions (cerebellum, amygdala, cortex, hippocampus, striatum, mesencephalon, medulla oblongata, substantia nigra and ventral tegmental area). Hence, this assay represents a valuable bioanalytical tool to support several pre(non)clinical studies in the broad field of neurosciences, requiring the quantitative analysis of these bioamines and their metabolites.