13C/15N‐Enriched L‐Dopa as a Triple‐Resonance NMR Probe to Monitor Neurotransmitter Dopamine in the Brain and Liver Extracts of Mice

In an attempt to monitor μM-level trace constituents, we applied here 1H-{13C-15N} triple-resonance nuclear magnetic resonance (NMR) to 13C/15N-enriched L-Dopa as the inevitable precursor of the neurotransmitter dopamine in the brain. The perfect selectivity (to render endogenous components silent) and μM-level sensitivity (700 MHz spectrometer equipped with a cryogenic probe) of triple-resonance allowed the unambiguous and quantitative metabolic and pharmacokinetic analyses of administered L-Dopa/dopamine in the brain and liver of mice. The level of dopamine generated in the brain (within the range 7–76 μM, which covers the typical stimulated level of ~30 μM) could be clearly monitored ex vivo, but was slightly short of the detection limit of a 7T MR machine for small animals. This work suggests that μM-level trace constituents are potential targets of ex vivo monitoring as long as they contain N atom(s) and their appropriate 13C/15N-enrichment is synthetically accessible

Intravenous administration of mesenchymal stem cells exerts therapeutic effects on parkinsonian model of rats: Focusing on neuroprotective effects of stromal cell-derived factor-1α

<p>Abstract</p> <p>Background</p> <p>Mesenchymal stem cells (MSCs) are pluripotent stem cells derived from bone marrow with secretory functions of various neurotrophic factors. Stromal cell-derived factor-1α (SDF-1α) is also reported as one of chemokines released from MSCs. In this research, the therapeutic effects of MSCs through SDF-1α were explored. 6-hydroxydopamine (6-OHDA, 20 μg) was injected into the right striatum of female SD rats with subsequent administration of GFP-labeled MSCs, fibroblasts, (i.v., 1 × 10⁷cells, respectively) or PBS at 2 hours after 6-OHDA injection. All rats were evaluated behaviorally with cylinder test and amphetamine-induced rotation test for 1 month with consequent euthanasia for immunohistochemical evaluations. Additionally, to explore the underlying mechanisms, neuroprotective effects of SDF-1α were explored using 6-OHDA-exposed PC12 cells by using dopamine (DA) assay and TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining.</p> <p>Results</p> <p>Rats receiving MSC transplantation significantly ameliorated behaviorally both in cylinder test and amphetamine-induced rotation test compared with the control groups. Correspondingly, rats with MSCs displayed significant preservation in the density of tyrosine hydroxylase (TH)-positive fibers in the striatum and the number of TH-positive neurons in the substantia nigra pars compacta (SNc) compared to that of control rats. In the <it>in vitro </it>study, SDF-1α treatment increased DA release and suppressed cell death induced by 6-OHDA administration compared with the control groups.</p> <p>Conclusions</p> <p>Consequently, MSC transplantation might exert neuroprotection on 6-OHDA-exposed dopaminergic neurons at least partly through anti-apoptotic effects of SDF-1α. The results demonstrate the potentials of intravenous MSC administration for clinical applications, although further explorations are required.</p

Neuroprotective effects of edaravone-administration on 6-OHDA-treated dopaminergic neurons

<p>Abstract</p> <p>Background</p> <p>Parkinson's disease (PD) is a neurological disorder characterized by the degeneration of nigrostriatal dopaminergic systems. Free radicals induced by oxidative stress are involved in the mechanisms of cell death in PD. This study clarifies the neuroprotective effects of edaravone (MCI-186, 3-methyl-1-phenyl-2-pyrazolin-5-one), which has already been used for the treatment of cerebral ischemia in Japan, on TH-positive dopaminergic neurons using PD model both <it>in vitro </it>and <it>in vivo</it>. 6-hydroxydopamine (6-OHDA), a neurotoxin for dopaminergic neurons, was added to cultured dopaminergic neurons derived from murine embryonal ventral mesencephalon with subsequet administration of edaravone or saline. The number of surviving TH-positive neurons and the degree of cell damage induced by free radicals were analyzed. In parallel, edaravone or saline was intravenously administered for PD model of rats receiving intrastriatal 6-OHDA lesion with subsequent behavioral and histological analyses.</p> <p>Results</p> <p><it>In vitro </it>study showed that edaravone significantly ameliorated the survival of TH-positive neurons in a dose-responsive manner. The number of apoptotic cells and HEt-positive cells significantly decreased, thus indicating that the neuroprotective effects of edaravone might be mediated by anti-apoptotic effects through the suppression of free radicals by edaravone. <it>In vivo </it>study demonstrated that edaravone-administration at 30 minutes after 6-OHDA lesion reduced the number of amphetamine-induced rotations significantly than edaravone-administration at 24 hours. Tyrosine hydroxylase (TH) staining of the striatum and substantia nigra pars compacta revealed that edaravone might exert neuroprotective effects on nigrostriatal dopaminergic systems. The neuroprotective effects were prominent when edaravone was administered early and in high concentration. TUNEL, HEt and Iba-1 staining <it>in vivo </it>might demonstrate the involvement of anti-apoptotic, anti-oxidative and anti-inflammatory effects of edaravone-administration.</p> <p>Conclusion</p> <p>Edaravone exerts neuroprotective effects on PD model both <it>in vitro and in vivo</it>. The underlying mechanisms might be involved in the anti-apoptotic effects, anti-oxidative effects, and/or anti-inflammatory effects of edaravone. Edaravone might be a hopeful therapeutic option for PD, although the high therapeutic dosage remains to be solved for the clinical application.</p

13C/15N-Enriched l -Dopa as a Triple-Resonance NMR Probe to Monitor Neurotransmitter Dopamine in the Brain and Liver Extracts of Mice

In an attempt to monitor μm-level trace constituents, we applied here 1H-{13C-15N} triple-resonance nuclear magnetic resonance (NMR) to 13C/15N-enriched l-Dopa as the inevitable precursor of the neurotransmitter dopamine in the brain. The perfect selectivity (to render endogenous components silent) and μm-level sensitivity (700 MHz spectrometer equipped with a cryogenic probe) of triple-resonance allowed the unambiguous and quantitative metabolic and pharmacokinetic analyses of administered l-Dopa/dopamine in the brain and liver of mice. The level of dopamine generated in the brain (within the range 7-76 μm, which covers the typical stimulated level of -30 μm) could be clearly monitored ex vivo, but was slightly short of the detection limit of a 7 T MR machine for small animals. This work suggests that μm-level trace constituents are potential targets of ex vivo monitoring as long as they contain N atom(s) and their appropriate 13C/15N-enrichment is synthetically accessible. Traces via triple resonance! Tripleresonance high-resolution NMR with a cryogenic probe at 16.4 T is sensitive and selective enough to perform quantitative metabolic/pharmacokinetic analysis of l-Dopa/dopamine in brain and liver extracts from mice. This work suggests that μm-level trace constituents are potential targets of ex vivo monitoring as long as they contain N atoms and their appropriate 13C/15N-enrichment is synthetically accessible