Development of a modified p-dimethylaminocinnamaldehyde solution for touch DNA analysis and its application to STR analysis.

We developed an acid-free p-dimethylaminocinnamaldehyde (DMAC) solution containing silicone oil that was suitable for spraying on clothing for analysis of biological samples such as touch DNA. We investigated the effect of this solution and irradiation with blue light emitting diode (LED) light on short tandem repeat (STR) analysis. To examine the effect of adding acid to the DMAC solution on visualizing biological samples, saliva sample was deposited on T-shirt. The T-shirt was sprayed with acid-added DMAC or acid-free DMAC solution and left for 2 h before irradiation with the blue LED light. We observed no differences between the fluorescence intensities achieved with these two solutions. To examine the effect of acid addition to the DMAC solution on STR analysis, sweat samples were smeared on glass slides and dried. The slides were sprayed with acid-added or acid-free DMAC solution and irradiated with the blue LED light. Samples were collected from the slides with swabs, and DNA was extracted from each sample using a PrepFiler Express™ Forensic DNA Extraction Kit and quantified using a Human DNA quantification kit (Takara RR281). The extracted DNA was amplified using an AmpFℓSTR® Identifiler® Plus PCR Amplification Kit for STR typing. We found that addition of acid to DMAC had little effect on DNA contained in the biological samples and STR analysis. To investigate whether DMAC could be used to visualize biological samples on clothes, saliva, sweat, and finger and palm prints were deposited on separate T-shirts. Biological samples were treated with DMAC and observed after 2 h, 1, 2, or 3 days under the blue LED. All biological samples were visualized and emitted fluorescence after 2 h. To examine the effects of the DMAC solution and LED irradiation on STR analysis, including DNA extraction, quantification, and STR typing, saliva and sweat were smeared on glass slides and dried. Touch DNA samples were deposited on glass slides directly. The slides were then sprayed with DMAC solution, sprayed with DMAC solution and irradiated with the blue LED, or left untreated. Samples were collected from the slides with swabs, and DNA was extracted, quantified, and amplified using the above kits. These results suggest that the DMAC solution and blue LED light will have no adverse effects on STR analysis. Therefore, this method will be very useful for touch DNA analysis in forensic investigations.

Therapeutic effect of a novel anti-parkinsonian agent zonisamide against MPTP (1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine) neurotoxicity in mice.

We investigated the therapeutic effect of zonisamide against 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice, using Western blot analysis, immunohistochemistry and behavioral test. Our Western blot analysis and immunohistochemical study showed that the post-treatment with zonisamide prevented significantly dopaminergic cell damage, the depletion of tyrosine-hydroxylase (TH) protein levels and the proliferation of microglia in the striatum and/or substantia nigra 8 days after MPTP treatment. Furthermore, our behavioral study showed that the post-treatment with zonisamide attenuated significantly the motor deficits 7 days after MPTP treatment. These results show that zonisamide has the therapeutic effect in the MPTP model of Parkinson's disease (PD) in mice. Our study also demonstrates the neuroprotective effect of zonisamide against dopaminergic cell damage after MPTP treatment in mice. Thus our present findings suggest that therapeutic strategies targeted to the activation of TH protein and/or the inhibition of microglial activation with zonisamide may offer a great potential for restoring the functional capacity of the surviving dopaminergic neurons in individuals affected with PD.

Therapeutic effect of a novel anti-parkinsonian agent zonisamide against MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) neurotoxicity in mice.

We investigated the therapeutic effect of zonisamide against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice, using Western blot analysis, immunohistochemistry and behavioral test. Our Western blot analysis and immunohistochemical study showed that the post-treatment with zonisamide prevented significantly dopaminergic cell damage, the depletion of tyrosine-hydroxylase (TH) protein levels and the proliferation of microglia in the striatum and/or substantia nigra 8 days after MPTP treatment. Furthermore, our behavioral study showed that the post-treatment with zonisamide attenuated significantly the motor deficits 7 days after MPTP treatment. These results show that zonisamide has the therapeutic effect in the MPTP model of Parkinson's disease (PD) in mice. Our study also demonstrates the neuroprotective effect of zonisamide against dopaminergic cell damage after MPTP treatment in mice. Thus our present findings suggest that therapeutic strategies targeted to the activation of TH protein and/or the inhibition of microglial activation with zonisamide may offer a great potential for restoring the functional capacity of the surviving dopaminergic neurons in individuals affected with PD.

Chronic administration with rotenone does not enhance MPTP neurotoxicity in C57BL/6 mice.

Systematic administration of rotenone as one of pesticides is known to produce degeneration of nigral dopaminergic neurons and motor deficits in experimental animals. Here, we investigated to determine whether systematic administration of rotenone causes the increased susceptibility in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Rotenone was injected into MPTP-treated mice over a period of 4 weeks. Thereafter, we evaluated the effect of rotenone 1, 3, and 6 weeks after the cessation of treatment with rotenone. In the present study with HPLC analysis, rotenone did not enhance MPTP-induced dopaminergic neurotoxicity in mice. Furthermore, MPTP + rotenone (9 mg/kg)-treated mice exhibit a significant loss of motor activity 1 day after the cessation of treatment with rotenone, However, no significant change of motor activity was found in MPTP-treated and MPTP + rotenone (9 mg/kg)-treated animals 6 weeks after the cessation of treatment with 0.5% carboxymethyl cellulose or rotenone. Our Western blot analysis study demonstrated that the change of tyrosine hydroxylase and glial fibrillary acidic protein protein levels in MPTP-treated mice was similar than that in MPTP + rotenone-treated animals. These results suggest that rotenone did not enhance MPTP neurotoxicity in mice. Our findings suggest that rotenone is not a reliable model for PD. Thus, our findings provide further valuable information for the pathogenesis of PD for exposure to agricultural pesticides.

Systemic administration of proteasome inhibitor protects against MPTP neurotoxicity in mice.

Dysfunction of the proteasome has been suggested to contribute in the degeneration of nigrostriatal dopaminergic neurons. Here, we investigated to determine whether systematic administration of proteasome inhibitor, carbobenzoxy-L: -gamma-t-butyl-L: -glutamyl-L: -alanyl-L: -leucinal (PSI) protects against MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) neurotoxicity in mice. Three administrations of MPTP at 1-h intervals to mice reduced significantly the concentration of dopamine, DOPAC (3,4-dihydroxyphenylacetic acid) and HVA (homovanillic acid) in the striatum after 5 days. In contrast, PSI (0.3 and 1.0 mg/kg) prevented a significant decrease in dopamine, DOPAC and HVA contents of the striatum 5 days after MPTP treatment. In our Western blot analysis study, PSI at a dose of 1.0 mg/kg prevented a significant decrease in TH (tyrosine hydroxylase) protein and a significant increase in glial fibrillary acidic protein 5 days after MPTP treatment. Furthermore, our immunohistochemical study showed that PSI at a dose of 1.0 mg/kg prevented a significant loss in TH immunopositive neurons in the striatum and substantia nigra 5 days after MPTP treatment. In contrast, PSI caused a significant increase in the number of intense ubiquitin immunopositive cells in the striatum and substantia nigra 5 days after MPTP treatment. These results indicate that proteasome inhibitors can protect against MPTP neurotoxicity in mice. The neuroprotective effect of PSI against dopaminergic cell damage may be mediated by the elevation of ubiquitination. Thus, our findings provide further valuable information for the pathogenesis of Parkinson's disease.

A novel anti-Parkinsonian agent, zonisamide, attenuates MPTP-induced neurotoxicity in mice.

Zonisamide, an anti-convulsant drug, has recently been shown to exert beneficial effects in Parkinson's disease (PD). However, actual pathophysiological mechanism underlying the anti-parkinsonian effect of zonisamide remains uncertain. Here we tested exactly the neuroprotective effect of zonisamide against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice. We observed that zonisamide attenuated MPTP-induced dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) depletion in the striatum and reduced the loss of tyrosine hidroxylase (TH) positive neurons and the increase of glial fibrillary acidic protein (GFAP) positive astrocytes in the striatum and substantia nigra after 5 days. Our Western blot analysis study also showed that zonisamide can prevent the decrease of TH protein levels and increase of GFAP protein levels in the striatum 5 days after MPTP treatment. In the present study, on the other hand, zonisaimde treatment showed no significant changes of the striatal dopamine, DOPAC, and HVA content in the striatum of normal mice after 1 day, as compared to the vehicle-treated group. Furthermore, zonisamide produced a significant increase of the TH protein levels in the striatum after 1 day, as compared to vehicle-treated group. In contrast, zonisamide showed no significant changes of the GFAP protein levels in the striatum after 1 day, as compared to vehicle-treated group. These results show that anticonvulsant drug, zonisamide, has the neuroprotective effect in the MPTP model of PD in mice. Our study also demonstrates that the neuroprotective effect of zonisamide against dopaminergic cell damage may be mediated by the elevation of TH activity on dopaminergic system after MPTP treatment in mice. Our findings suggest that zonisamide may offer a new approach for the treatment of PD.

Role of nuclear transcription factor kappa B (NF-kappaB) for MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine)-induced apoptosis in nigral neurons of mice.

The biochemical and cellular changes that occur following treatment with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine) are remarkably similar to that seen in idiopathic Parkinson's disease. In this study, we investigated the time course changes of NF-kappaB (Nuclear factor kappa B) p65 protein and apoptosis in the substantia nigra after MPTP treatment in mice. Four administrations of MPTP at 2 h intervals showed a significant and severe decrease of the number of TH (tyrosine hydroxylase) immunopositive neurons in the substantia nigra of mice from 5 h up to 21 days posttreatment. Densities of DAT (dopamine transporter) immunoreactivity were also significantly decreased in nigral neurons of mice from 1 up to 21 days after MPTP treatment. GFAP (glial fibrillary acidic protein) immunopositive cells were increased significantly in the substantia nigra from 5 h up to 21 days after MPTP treatment. In contrast, isolectin B(4) positive microglia were increased markedly in the substantia nigra only 3 and 7 days after MPTP treatment. On the other hand, a significant increase of NF-kappaB p65 immunoreactivity was observed mainly in glial cells of the substantia nigra from 5 h to 3 days after MPTP treatment. A significant increase of ssDNA (single stranded DNA) immunopositive apoptotic neurons was also observed in the substantia nigra from 5 h to 3 days after MPTP treatment. These results demonstrate that dopaminergic neuronal loss may be caused by apoptosis due to increased cytokines and apoptosis-related proteins via the activation of NF-kappaB in reactive astrocytes of the substantia nigra after MPTP treatment in mice. Thus our findings suggest that the inhibition of NF-kappaB activation in astrocytes may be useful intervention in Parkinson's disease and other neurogenerative disorders where apoptosis or inflammation plays a key role in disease pathogenesis.

Targeting reactive oxygen species, reactive nitrogen species and inflammation in MPTP neurotoxicity and Parkinson's disease.

Parkinson's disease (PD) is the second most frequent neurodegenerative disorder after Alzheimer's disease. The main clinical features of PD include tremor, bradykinesia, rigidity and postural instability. The primary pathology of PD is degeneration of dopaminergic neurons in the substantia nigra pars compacta, resulting in loss of the nigrostriatal pathway and a reduction of dopamine contents in the striatum. The biochemical and cellular changes that occur following the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are remarkably similar to that seen in idiopathic PD. Recent evidence shows that oxidative stress contributes to the cascade leading to dopaminergic cell degeneration in PD. However, oxidative stress is intimately linked to other components of neurodegenerative process, such as nitric oxide stress and inflammation. Recently, there is convincing evidence for the involvement of nitric oxide that reacts with superoxide to produce peroxynitrite and ultimately hydroxyl radical production. In view of these new insights, however, the role of reactive nitrogen species, reactive oxygen species and inflammation against MPTP neurotoxicity is not fully understood. In this review, we discuss the possible role of reactive nitrogen species, reactive oxygen species and inflammation in the dopaminergic neurons against MPTP neurotoxicity.

Comparative pharmacological study of free radical scavenger, nitric oxide synthase inhibitor, nitric oxide synthase activator and cyclooxygenase inhibitor against MPTP neurotoxicity in mice.

The biochemical and cellular changes that occur following the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are remarkably similar to that seen in idiopathic Parkinson's disease(PD). There is growing evidence indicating that reactive oxygen species (ROS), reactive nitrogen species (RNS) and inflammation are a major contributor to the pathogenesis and progression of PD. Hence, we investigated whether 7-nitroindazole [neuronal nitric oxide synthase (nNOS) inhibitor], edaravone (free radical scavenger), minocycline [inducible NOS (iNOS) inhibitor], fluvastatin [endothelial NOS (eNOS) activator], pitavastatin (eNOS activator), etodolac [cyclooxygenase-2 (COX-2) inhibitor] and indomethacin (COX inhibitor) can protect against MPTP neurotoxicity in mice under the same conditions. For the evaluation of each drug, the levels of dopamine, DOPAC and HVA were quantified using HPLC with an electrochemical detector. Four administrations of MPTP at 1-h intervals to mice produced marked depletion of dopamine, DOPAC (3,4-dihydroxyphenylacetic acid) and HVA (homovanilic acid) in the striatum after 5 days. 7-Nitroindazole prevented dose-dependently a significant reduction in dopamine contents of the striatum 5 days after MPTP treatment. In contrast, edaravone, minocycline, fluvastatin, pitavastatin, etodolac and indomethacin did not show the neuroprotective effect on MPTP-induced striatal dopamine, DOPAC and HVA depletions after 5 days. The present study demonstrates that the overexpression of nNOS may play a major role in the neurotoxic processes of MPTP, as compared with the production of ROS, the overexpression of iNOS, the modulation of eNOS and the involvement of inflammatory response. Thus our pharmacological findings provide further information for progressive neurodegeneration of the nigrostriatal dopaminergic neuronal pathway.

Failure of acute administration with proteasome inhibitor to provide a model of Parkinson's disease in mice.

We investigated to determine whether acute administration of proteasome inhibitor can cause dopaminergic cell loss in mice, in comparison with that of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The four intraperitoneally administrations of MPTP at 1-h intervals to mice decreased significantly the concentration of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum after 5 days, in comparison with vehicle-treated animals. In contrast, the three subcutaneously administrations of carbobenzoxy-L-gamma-t-butyl-L-glutamyl-L-alanyl-L-leucinal (PSI) did not show significant changes in the concentration of dopamine, DOPAC and HVA in the striatum after 5 days, in comparison with vehicle-treated animals. Our Western blot analysis also showed that the four administrations of MPTP at 1-h intervals to mice produced a significant reduction of anti-tyrosine hydroxylase antibody (TH) protein levels in the striatum after 5 days after. In PSI-treated mice. In contrast, no significant change of TH protein levels was observed in the striatum 5 days after the final treatment with PSI. Furthermore, a significant decrease of TH protein levels was observed in the striatum of MPTP-treated mice, as compared with PSI-treated animals. The present study demonstrates that the acute treatment with proteasome inhibitor PSI did not cause the dopaminergic neurotoxicity in mice, as compared with acute treatment with MPTP. Thus, our findings suggest that acute proteasome inhibition is not a reliable model for Parkinson's disease.