Preventive effects of nano-graphene oxide against Parkinson's disease via reactive oxygen species scavenging and anti-inflammation.

We investigated the neuroprotective effects of deca nano-graphene oxide (daNGO) against reactive oxygen species (ROS) and inflammation in the human neuroblastoma cell line SH-SY5Y and in the 6-hydroxydopamine (6-OHDA) induced Parkinsonian rat model. An MTT assay was performed to measure cell viability in vitro in the presence of 6-OHDA and/or daNGO. The intracellular ROS level was quantified using 2',7'-dichlorofluorescein diacetate. daNGO showed neuroprotective effects against 6-OHDA-induced toxicity and also displayed ROS scavenging properties. We then tested the protective effects of daNGO against 6-OHDA induced toxicity in a rat model. Stepping tests showed that the akinesia symptoms were improved in the daNGO group compared to the control group. Moreover, in an apomorphine-induced rotation test, the number of net contralateral rotations was decreased in the daNGO group compared to the control group. By immunofluorescent staining, the animals in the daNGO group had more tyrosine hydroxylase-positive cells than the controls. By anti-Iba1 staining, 6-OHDA induced microglial activation showed a significantly decrease in the daNGO group, indicating that the neuroprotective effects of graphene resulted from anti-inflammation. In conclusion, nanographene oxide has neuroprotective effects against the neurotoxin induced by 6-OHDA on dopaminergic neurons. [BMB Reports 2023; 56(3): 202-207].

CRISPR-Cas9 Gene Editing Protects from the A53T-SNCA Overexpression-Induced Pathology of Parkinson's Disease In Vivo.

Mutations in specific genes, including synuclein alpha (SNCA) that encodes the α-synuclein protein, are known to be risk factors for sporadic Parkinson's disease (PD), as well as critical factors for familial PD. In particular, A53T-mutated SNCA (A53T-SNCA) is a well-studied familial pathologic mutation in PD. However, techniques for deletion of the mutated SNCA gene in vivo have not been developed. Here, we used the CRISPR-Cas9 system to delete A53T-SNCA in vitro as well as in vivo. Adeno-associated virus carrying SaCas9-KKH with a single-guide RNA targeting A53T-SNCA significantly reduced A53T-SNCA expression levels in vitro. Furthermore, we tested its therapeutic potential in vivo in a viral A53T-SNCA-overexpressing rat model of PD. Gene deletion of A53T-SNCA significantly rescued the overexpression of α-synuclein, reactive microgliosis, dopaminergic neurodegeneration, and parkinsonian motor symptoms. Our findings propose CRISPR-Cas9 system as a potential prevention strategy for A53T-SNCA-specific PD.

The risk factors of intracerebral hemorrhage in deep brain stimulation: does target matter?

BACKGROUND: Although deep brain stimulation (DBS) is a relatively safe and effective surgery compared with ablative surgeries, intracerebral hemorrhage (ICH) is a serious complication during DBS that could result in a fatal prognosis. We retrospectively investigated whether ICH incidence differed between patients who underwent DBS in the subthalamic nucleus (STN) and in the globus pallidus interna (GPi), together with previously identified risk factors for ICH. METHODS: We retrospectively reviewed the medical records of 275 patients (527 DBS targets) who received DBS for Parkinson's disease or dystonia from April 2001 to December 2020. In cases that developed intra- or postoperative ICH, patients were classified as asymptomatic, symptomatic with temporary neurological deficit or symptomatic with permanent neurological deficit, according to patient clinical status. RESULTS: ICH occurred in 12 procedures (2.3%) among the 527 DBS procedures (275 patients) evaluated. In multivariable logistic regression analysis, the risk factor for all cases of ICH was systolic blood pressure (BP) during surgery (cut-off value 129.4 mmHg) (OR = 1.05, 95% CI = 1.01-1.09, P = 0.023). In addition, for ICH with permanent neurological deficit, STN target site (P = 0.024) and systolic BP during surgery (cut-off value: 148.3 mmHg) (P = 0.004) were identified as risk factors in univariable analyses. CONCLUSION: Even though the risk factor for all ICH in DBS was BP during surgery, when focused on ICH evoking permanent neurological deficit, the target location as well as systolic BP during surgery proved to be related.

KDS2010, a Newly Developed Reversible MAO-B Inhibitor, as an Effective Therapeutic Candidate for Parkinson's Disease.

Monoamine oxidase-B (MAO-B) is a well-established therapeutic target for Parkinson's disease (PD); however, previous clinical studies on currently available irreversible MAO-B inhibitors have yielded disappointing neuroprotective effects. Here, we tested the therapeutic potential of KDS2010, a recently synthesized potent, selective, and reversible MAO-B inhibitor in multiple animal models of PD. We designed and synthesized a series of α-aminoamide derivatives and found that derivative KDS2010 exhibited the highest potency, specificity, reversibility, and bioavailability (> 100%). In addition, KDS2010 demonstrated significant neuroprotective and anti-neuroinflammatory efficacy against nigrostriatal pathway destruction in the mouse MPTP model of parkinsonism. Treatment with KDS2010 also alleviated parkinsonian motor dysfunction in 6-hydroxydopamine-induced and A53T mutant α-synuclein overexpression rat models of PD. Moreover, KDS2010 showed virtually no toxicity or side effects in non-human primates. KDS2010 could be a next-generation therapeutic candidate for PD.

Optimal Ratio of Wnt3a Expression in Human Mesenchymal Stem Cells Promotes Axonal Regeneration in Spinal Cord Injured Rat Model.

OBJECTIVE: Through our previous clinical trials, the demonstrated therapeutic effects of MSC in chronic spinal cord injury (SCI) were found to be not sufficient. Therefore, the need to develop stem cell agent with enhanced efficacy is increased. We transplanted enhanced Wnt3asecreting human mesenchymal stem cells (hMSC) into injured spines at 6 weeks after SCI to improve axonal regeneration in a rat model of chronic SCI. We hypothesized that enhanced Wnt3a protein expression could augment neuro-regeneration after SCI. METHODS: Thirty-six Sprague-Dawley rats were injured using an Infinite Horizon (IH) impactor at the T9-10 vertebrae and separated into five groups : 1) phosphate-buffered saline injection (injury only group, n=7); 2) hMSC transplantation (MSC, n=7); 3) hMSC transfected with pLenti vector (without Wnt3a gene) transplantation (pLenti-MSC, n=7); 4) hMSC transfected with Wnt3a gene transplantation (Wnt3a-MSC, n=7); and 5) hMSC transfected with enhanced Wnt3a gene (1.7 fold Wnt3a mRNA expression) transplantation (1.7 Wnt3a-MSC, n=8). Six weeks after SCI, each 5×105 cells/15 µL at 2 points were injected using stereotactic and microsyringe pump. To evaluate functional recovery from SCI, rats underwent Basso-Beattie-Bresnahan (BBB) locomotor test on the first, second, and third days post-injury and then weekly for 14 weeks. Axonal regeneration was assessed using growth-associated protein 43 (GAP43), microtubule-associated protein 2 (MAP2), and neurofilament (NF) immunostaining. RESULTS: Fourteen weeks after injury (8 weeks after transplantation), BBB score of the 1.7 Wnt3a-MSC group (15.0±0.28) was significantly higher than that of the injury only (10.0±0.48), MSC (12.57±0.48), pLenti-MSC (12.42±0.48), and Wnt3a-MSC (13.71±0.61) groups (p<0.05). Immunostaining revealed increased expression of axonal regeneration markers GAP43, MAP2, and NF in the Wnt3a-MSC and 1.7 Wnt3a-MSC groups. CONCLUSION: Our results showed that enhanced gene expression of Wnt3a in hMSC can potentiate axonal regeneration and improve functional recovery in a rat model of chronic SCI.

Optogenetic inactivation of the entopeduncular nucleus improves forelimb akinesia in a Parkinson's disease model.

We performed optogenetic inactivation of rats' entopeduncular nucleus (EP, homologous to primates' globus pallidus interna (GPi)) and investigated the therapeutic effect in a rat model of PD. 6-Hydroxydopamine (6-OHDA)-induced hemiparkinsonian rats were injected with either a virus for halorhodopsin expression that is used to inactivate GABAergic neurons or a control virus injection and received optic fiber insertion. All the rats were illuminated by 590 nm of light. Each rat was then subjected to sequential sessions of stepping tests under controlled illumination patterns. The stepping test is a reliable evaluation method for forelimb akinesia. The number of adjusting steps was significantly higher in experimental (optogene with reporter gene expression) (5Hz - 10ms: 15.7 ±â€¯1.9, 5Hz - 100ms: 16.0 ±â€¯1.8, continuous: 21.6 ±â€¯1.9) than control rats (reporter gene expression) (5Hz-10ms: 1.9 ±â€¯1.1, 5Hz-100ms: 2.6 ±â€¯1.0, continuous: 2.5 ±â€¯1.2) (p < 0.001). Continuous EP illumination showed a significantly higher improvement of forelimb akinesia than other illumination patterns (p < 0.01). Optogene expression in the GABAergic neurons of the EP was confirmed by immunohistochemistry. Optogenetic inhibition of EP was effective to improve contralateral forelimb akinesia. However, further studies using prolonged illumination are needed to investigate the best illumination pattern for optogenetic stimulation.

Aberrant Tonic Inhibition of Dopaminergic Neuronal Activity Causes Motor Symptoms in Animal Models of Parkinson's Disease.

Current pharmacological treatments for Parkinson's disease (PD) are focused on symptomatic relief, but not on disease modification, based on the strong belief that PD is caused by irreversible dopaminergic neuronal death. Thus, the concept of the presence of dormant dopaminergic neurons and its possibility as the disease-modifying therapeutic target against PD have not been explored. Here we show that optogenetic activation of substantia nigra pars compacta (SNpc) neurons alleviates parkinsonism in acute PD animal models by recovering tyrosine hydroxylase (TH) from the TH-negative dormant dopaminergic neurons, some of which still express DOPA decarboxylase (DDC). The TH loss depends on reduced dopaminergic neuronal firing under aberrant tonic inhibition, which is attributed to excessive astrocytic GABA. Blocking the astrocytic GABA synthesis recapitulates the therapeutic effect of optogenetic activation. Consistently, SNpc of postmortem PD patients shows a significant population of TH-negative/DDC-positive dormant neurons surrounded by numerous GABA-positive astrocytes. We propose that disinhibiting dormant dopaminergic neurons by blocking excessive astrocytic GABA could be an effective therapeutic strategy against PD.

A Novel Animal Model of Parkinson's Disease Using Optogenetics: Representation of Various Disease Stages by Modulating the Illumination Parameter.

BACKGROUND: The classic animal model of Parkinson's disease (PD) using neurotoxin can only simulate fixed stages of the disease by causing irreversible damage to the nigrostriatal system. OBJECTIVES: To develop an optogenetic PD model that can modulate the severity of disease by optical stimulation by introducing the halorhodopsin (NpHR) gene into the substantia nigra compacta. METHODS: Fifteen rats received injections of engineered AAV with NpHR-YFP gene into the substantia nigra. They were then subjected to illumination of 590-nm light wavelengths with 3 optical stimulation conditions, i.e., frequency-width: 5 Hz-10 ms (n = 5), 5 Hz-100 ms (n = 5), and 50 Hz-10 ms (n = 5). Eleven rats received 6-hydroxydopamine injections to establish the conventional PD model. RESULTS: The optogenetic models showed characteristic PD manifestations, similar to those of the conventional models; the severity of forelimb akinesia correlated with the total illumination value (frequency × width). The group with a low illumination value (5 Hz-10 ms) was comparable to the conventional partial model whereas the groups with high illumination values (5 Hz-100 ms and 50 Hz-10 ms) were similar to the conventional complete model. CONCLUSIONS: An optogenetic PD model has the advantage of more appropriately representing various PD stages by controlling illumination parameters.

Is There Additive Therapeutic Effect When GCSF Combined with Adipose-Derived Stem Cell in a Rat Model of Acute Spinal Cord Injury?

OBJECTIVE: Functional and neural tissue recovery has been reported in many animal studies conducted with stem cells. However, the combined effect of cytokines and stem cells has not yet been adequately researched. Here, we analyzed the additive effects of granulocyte colony-stimulating factor (GCSF) on adipose-derived stem cells (ADSCs) infusion in the treatment of acute spinal cord injury (SCI) in rats. METHODS: Four days after intrathecal infusion tubes implantation in Sprague-Dawley rats, SCI was induced with an infinite horizon impactor. In the Sham group (n=5), phosphate-buffered saline was injected 3, 7, and 14 days after SCI. GCSF, ADSCs, and ADSCs with GCSF were injected at the same time in the GCSF (n=8), ADSC (n=8), and ADSC+GCSF groups (n=7), respectively. RESULTS: The ADSC and ADSC+GCSF groups, but not the GCSF group, showed significantly higher Basso-Beattie-Bresnahan scores than the Sham group during 8 weeks (p<0.01), but no significant difference between the ADSC and ADSC+GCSF groups. In the ladder rung test, all four groups were significantly different from each other, with the ADSC+GCSF group showing the best improvement (p<0.01). On immunofluorescent staining (GAP43, MAP2), western blotting (GAP43), and reverse transcription polymerase chain reaction (GAP43, nerve growth factor), the ADSC and ADSC+GCSF groups showed higher levels than the Sham and GCSF groups. CONCLUSION: Our analyses suggest that the combination of GCSF and ADSCs infusions in acute SCI in the rat does not have a significant additive effect. Hence, when combination agents for SCI stem cell therapy are considered, molecules other than GCSF, or modifications to the methodology, should be investigated.

The Rate of Drug-Resistant Tuberculosis in Korean Children and Adolescents Since 2007.

The incidence of drug-resistant tuberculosis (DR-TB) in pediatric populations is a critical indicator of national TB management and treatment strategies. Limited data exist regarding the rate of pediatric DR-TB. In this study, we aimed to analyze the status of DR-TB in Korean children from 2007 to 2013. We analyzed specimens submitted to the Korean Institute of Tuberculosis using Mycobacterium tuberculosis culture and drug susceptibility tests (DSTs) from January 2007 through December 2013. Specimens from patients ≤ 19 years of age were included. Among the 2,690 cases, 297 cases were excluded because of insufficient data, leaving 2,393 cases for the final analysis. In total, resistance to one or more TB drugs was 13.5%. The resistance rates of each of the drugs were as follows: isoniazid (INH) 10.2%, rifampin (RFP) 5.1%, ethambutol (EMB) 3.7%, and pyrazinamide (PZA) 3.1%. The resistance rate of multidrug-resistant TB (MDR-TB) was 4.2%, and that of extensively drug-resistant TB (XDR-TB) was 0.8%. The overall drug resistance rate demonstrated significant increase throughout the study period (P < 0.001) but showed no significant difference compared to previous study from 1999 to 2007. The drug resistance rate of PZA in ≤ 15 years of age group was significantly greater than that of > 15 years (P < 0.001). The drug resistance rate has increased throughout the study period.

Enhanced axonal regeneration by transplanted Wnt3a-secreting human mesenchymal stem cells in a rat model of spinal cord injury.

BACKGROUND: While pure mesenchymal stem cell (MSC) treatment for spinal cord injury (SCI) is known to be safe, its efficacy is insufficient. Therefore, gene-modified stem cells are being developed to enhance the effect of pure MSCs. We investigated the effect of stem cell therapy through the transfection of a Wnt3a-producing gene that stimulates axonal regeneration. METHOD: MSCs obtained from the human umbilical cord blood (hMSCs) were multiplied, cultivated, and transfected with the pLenti-Wnt3a-GFP viral vector to produce Wnt3a-secreting hMSCs. A total of 50 rats were injured with an Infinite Horizon impactor at the level of the T7-8 vertebrae. Rats were divided into five groups according to the transplanted material: (1) phosphate-buffered saline injection group (sham group, n = 10); (Pertz et al. Proc Natl Acad Sci USA 105:1931-1936, 39) Wnt3a protein injection group (Wnt3a protein group, n = 10); (3) hMSC transplantation group (MSC group, n = 10); (4) hMSCs transfected with the pLenti vector transplantation group (pLenti-MSC group, n = 10); (5) hMSCs transfected with the pLenti+Wnt3a vector transplantation group (Wnt3a-MSC group, n = 10). Behavioral tests were performed daily for the first 3 days after injury and then weekly for 8 weeks. The injured spinal cords were extracted, and axonal regeneration markers including choline acetyltransferase (ChAT), growth-associated protein 43 (GAP43), and microtubule-associated protein 2 (MAP2) were investigated by immunofluorescence, RT-PCR, and western blotting. RESULTS: Seven weeks after the transplantation (8 weeks after SCI), rats in the Wnt3a-MSC group achieved significantly higher average scores in the motor behavior tests than those in the other groups (p < 0.05). Immunofluorescent stains showed greater immunoreactivity of ChAT, GAP43, and MAP2 in the Wnt3a-MSC group than in the other groups. RT-PCR and western blots revealed greater expression of these proteins in the Wnt3a-MSC group than in the other groups (p < 0.05). CONCLUSIONS: Wnt3a-secreting hMSC transplantation considerably improved neurological recovery and axonal regeneration in a rat SCI model.

Optogenetic Inhibition of the Subthalamic Nucleus Reduces Levodopa-Induced Dyskinesias in a Rat Model of Parkinson's Disease.

BACKGROUND: The inhibition of neuronal activity by electrical deep brain stimulation is one of the mechanisms explaining the amelioration of levodopa-induced dyskinesia. However, electrical deep brain stimulation cannot specifically activate or inactivate selected types of neurons. OBJECTIVES: We applied optogenetics as an alternative treatment to deep brain stimulation for levodopa-induced dyskinesia, and also to confirm that the mechanism of levodopa-induced dyskinesia amelioration by subthalamic nucleus deep brain stimulation is mediated through neuronal inhibition. METHODS: 6-hydroxydopamine-induced hemiparkinsonian rats received injections of hSynapsin1-NpHR-YFP adeno-associated virus (AAV) or hSynapsin1-YFP AAV. Two weeks after viral injections, all rats were treated with daily injections of levodopa. Then, the optic fiber was implanted into the ipsilateral subthalamic nucleus. We performed various behavioral tests to evaluate the changes in levodopa-induced dyskinesias after optogenetic expression and illumination in the subthalamic nucleus. RESULTS: The behavioral tests revealed that optical inhibition of the subthalamic nucleus significantly ameliorated levodopa-induced dyskinesia by reducing the duration of the dyskinesias as well as the severity of axial dyskinesia. CONCLUSIONS: These findings will provide a useful foundation for the future development of optogenetic modulation systems that could be considered as an approach to dyskinesia therapy.

A rat model of striatonigral degeneration generated by simultaneous injection of 6-hydroxydopamine into the medial forebrain bundle and quinolinic acid into the striatum.

A double toxin-double lesion strategy is well-known to generate a rat model of striatonigral degeneration (SND) such as multiple system atrophy-parkinsonian type. However, with this model it is difficult to distinguish SND from Parkinson's disease (PD). In this study, we propose a new rat model of SND, which is generated by simultaneous injection of 6-hydroxydopamine into the medial forebrain bundle and quinolinic acid into the striatum. Stepping tests performed 30 min after intraperitoneal L-dopa administration at 6 weeks post-surgery revealed an L-dopa response in the PD group but not the SND group. Apomorphine-induced rotation tests revealed no rotational bias in the SND group, which persisted for 2 months, but contralateral rotations in the PD group. MicroPET scans revealed glucose hypometabolism and dopamine transporter impairment on the lesioned striatum in the SND group. Tyrosine hydroxylase immunostaining in the SND group revealed that 74.7% of nigral cells on the lesioned side were lost after lesion surgery. These results suggest that the proposed simultaneous double toxin-double lesion method successfully created a rat model of SND that had behavioral outcomes, multitracer microPET evaluation, and histological aspects consistent with SND pathology. This model will be useful for future study of SND.

Optogenetic inactivation of the subthalamic nucleus improves forelimb akinesia in a rat model of Parkinson disease.

BACKGROUND: The inhibition of neuronal activity by electrical deep brain stimulation is one of the mechanisms explaining the therapeutic effects in patients with Parkinson disease (PD) but cannot specifically activate or inactivate different types of neurons. Recently, a new technology based on optogenetics has been developed to modulate the activity of specific neurons. However, the therapeutic effects of optical inactivation in the subthalamic nucleus (STN) have not been fully investigated. OBJECTIVE: To perform various behavioral tests to evaluate changes in motor functions in a PD rat model after optogene expression and, unlike previous studies, to assess the therapeutic effects of direct optogenetic inactivation in the STN. METHODS: 6-Hydroxydopamine-induced hemiparkinsonian rats received injections of hSynapsin1-NpHR-YFP adeno-associated virus or an equivalent volume of phosphate-buffered saline. Three weeks after injection of adeno-associated virus or phosphate-buffered saline, the optic fiber was implanted into the ipsilateral STN. A stepping test, a cylinder test, and an apomorphine-induced rotation test were performed in 3 sequential steps: during light-off state, during light stimulation, and again during light-off state. RESULTS: Stepping tests revealed that optical inhibition of the STN significantly improved 6-hydroxydopamine-induced forelimb akinesia. PD motor signs, as assessed by cylinder and apomorphine tests, were not affected by optical inhibition. Immunofluorescence revealed that halorhodopsin was highly expressed and colocalized with vesicular glutamate transporter 2 in the STN. CONCLUSION: Optogenetic inhibition in the STN may be effective in improving contralateral forelimb akinesia but not in changing forelimb preference or reducing dopaminergic receptor supersensitivity. These findings are useful as a basis for future studies on optogenetics in PD.

Are human dental papilla-derived stem cell and human brain-derived neural stem cell transplantations suitable for treatment of Parkinson's disease?

Transplantation of neural stem cells has been reported as a possible approach for replacing impaired dopaminergic neurons. In this study, we tested the efficacy of early-stage human dental papilla-derived stem cells and human brain-derived neural stem cells in rat models of 6-hydroxydopamine-induced Parkinson's disease. Rats received a unilateral injection of 6-hydroxydopamine into right medial forebrain bundle, followed 3 weeks later by injections of PBS, early-stage human dental papilla-derived stem cells, or human brain-derived neural stem cells into the ipsilateral striatum. All of the rats in the human dental papilla-derived stem cell group died from tumor formation at around 2 weeks following cell transplantation. Postmortem examinations revealed homogeneous malignant tumors in the striatum of the human dental papilla-derived stem cell group. Stepping tests revealed that human brain-derived neural stem cell transplantation did not improve motor dysfunction. In apomorphine-induced rotation tests, neither the human brain-derived neural stem cell group nor the control groups (PBS injection) demonstrated significant changes. Glucose metabolism in the lesioned side of striatum was reduced by human brain-derived neural stem cell transplantation. [(18)F]-FP-CIT PET scans in the striatum did not demonstrate a significant increase in the human brain-derived neural stem cell group. Tyrosine hydroxylase (dopaminergic neuronal marker) staining and G protein-activated inward rectifier potassium channel 2 (A9 dopaminergic neuronal marker) were positive in the lesioned side of striatum in the human brain-derived neural stem cell group. The use of early-stage human dental papilla-derived stem cells confirmed its tendency to form tumors. Human brain-derived neural stem cells could be partially differentiated into dopaminergic neurons, but they did not secrete dopamine.

Evaluation of a multiple system atrophy model in rats using multitracer microPET.

BACKGROUND: A double toxin-double lesion strategy is appropriate for mimicking of striatonigral degeneration. Because knowledge of human pathology is limited, animal models must be well characterized prior to testing of therapeutic approaches to treat multiple system atrophy. In double-toxin animal models, however, reduced contralateral rotation after apomorphine injection is restored within a few weeks via an unknown mechanism; the animals thus revert to PD status. We assessed this phenomenon using multitracer microPET and tissue staining. METHODS: Five adult male Wistar rats received injections of 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle (MFB), followed 3 weeks later by injections of quinolinic acid (QA) into the ipsilateral striatum. Apomorphine-induced rotation tests were performed 1 week after each injection, and 6 and 10 weeks after QA injection. Rotarod tests were performed weekly after 6-OHDA injection. MSA-p status was characterized by microPET 5 and 10 weeks after QA injection using the tracers 2-deoxy-2-[(18)F]-fluoro-D-glucose ([(18)F]-FDG) and [(18)F]-N-(3-fluoropropyl)-2-carbomethoxy-3-(4-iodophenyl)nortropane ([(18)F]-FP-CIT). Histological changes were evaluated by tyrosine hydroxylase (TH) and cresyl violet staining. RESULTS: The numbers of apomorphine-induced rotations increased contralaterally after 6-OHDA lesions were created, but decreased significantly after QA administration (p = 0.007). Five weeks after QA injection, however, contralateral rotation again increased and persisted for 1 month. Rotarod rotation differed significantly between the intact and PD states (p < 0.05), but not between the PD and MSA-p states. MicroPET revealed glucose hypometabolism and dopamine transporter (DAT) impairment on the lesioned side of the striatum 1 and 2 months after QA lesion surgery. Loss of nigral cells was confirmed by TH immunostaining, and striatal atrophy was observed upon cresyl violet staining. CONCLUSION: Pathological changes consistent with MSA-p can be generated by the double toxin-double lesion method and persist during follow-up. Behavioral tests, such as drug-induced rotation and rotarod tests, are not appropriate for long-term follow-up in the MSA-p model, suggesting the need for development of more appropriate behavioral tests.