AMPK-mediated autophagy pathway activation promotes ΔFosB degradation to improve levodopa-induced dyskinesia.

BACKGROUND: Parkinson's disease patients on chronic levodopa often suffer from motor complications, which tend to reduce their quality of life. Levodopa-induced dyskinesia (LID) is one of the most prevalent motor complications, often characterized by abnormal involuntary movements, and the pathogenesis of LID is still unclear but recent studies have suggested the involvement of autophagy. METHODS: The onset of LID was mimicked by chronic levodopa treatment in a unilateral 6-hydroxydopamine (6-OHDA) -lesion rat model. Overexpression of ΔFosB in HEK293 cells to mimic the state of ΔFosB accumulation. The modulation of the AMP-activated protein kinase (AMPK)-mediated autophagy pathway using by metformin, AICAR (an AMPK activator), Compound C (an AMPK inhibitor) and chloroquine (an autophagy pathway inhibitor). The severity of LID was assessed by axial, limb, and orofacial (ALO) abnormal involuntary movements (AIMs) score and in vivo electrophysiology. The activity of AMPK pathway as well as autophagy markers and FosB-ΔFosB levels were detected by western blotting. RT-qPCR was performed to detect the transcription level of FosB-ΔFosB. The mechanism of autophagy dysfunction was further explored by immunofluorescence and transmission electron microscopy. RESULTS: In vivo experiments demonstrated that chronic levodopa treatment reduced AMPK phosphorylation, impaired autophagosome-lysosomal fusion and caused FosB-ΔFosB accumulation in the striatum of PD rats. Long-term metformin intervention improved ALO AIMs scores as well as reduced the mean power of high gamma (hγ) oscillations and the proportion of striatal projection neurons unstable in response to dopamine for LID rats. Moreover, the intervention of metformin promoted AMPK phosphorylation, ameliorated the impairment of autophagosome-lysosomal fusion, thus, promoting FosB-ΔFosB degradation to attenuate its accumulation in the striatum of LID rats. However, the aforementioned roles of metformin were reversed by Compound C and chloroquine. The results of in vitro studies demonstrated the ability of metformin and AICAR to attenuate ΔFosB levels by promoting its degradation, while Compound C and chloroquine could block this effect. CONCLUSIONS: In conclusion, our results suggest that long-term metformin treatment could promote ΔFosB degradation and thus attenuate the development of LID through activating the AMPK-mediated autophagy pathway. Overall, our results support the AMPK-mediated autophagy pathway as a novel therapeutic target for LID and also indicate that metformin is a promising therapeutic candidate for LID.

Therapeutic Effects of Selenium on Alpha-Synuclein Accumulation in Substantia Nigra Pars Compacta in a Rat Model of Parkinson's Disease: Behavioral and Biochemical Outcomes.

Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder characterized by the accumulation of accumulated alpha-synuclein (α-Syn) in substantia nigra. Research has shown that selenium (Se) can protect neural cells through the actions of selenoproteins, including selenoprotein P (SelP) and selenoprotein S (SelS), which participate in endoplasmic reticulum-associated protein degradation (ERAD). In this study, we investigated the potential protective role of Se in a pre-clinical PD rat model.We aimed to evaluate the therapeutic effects of Se administration in the 6-hydroxydopamine (6-OHDA) induced unilateral rat PD model. Male Wistar rats were utilised for unilateral PD animal model which were subjected to stereotaxic surgery and injected with 20 µg 6-OHDA/5 µl 0.2% ascorbate saline. After confirming the model, the rats were intraperitoneally injected with 0.1, 0.2, and 0.3 mg/kg of sodium selenite for 7 days. We then performed behavioral tests, including apomorphine-induced rotation, hanging, and rotarod tests. Following sacrifice, we analysed the substantia nigra area of the brain and serum for protein quantification, element analysis, and gene expression analysis.Our results indicate that the administration of 0.3 mg/kg of Se improved the motor deficiency in hanging, rotarod, and apomorphine-induced rotational tests. While there was no significant improvement in the expression of α-Syn, Se increased the expression of selenoproteins. Additionally, levels of selenoproteins, Se, and α-Syn both brain and serum were re-established by the treatment, suggesting the role of Se on the α-Syn accumulation. Furthermore, Se improved PD-induced biochemical deficits by increasing the levels of SelS and SelP (p<0.005).In conclusion, our findings suggest that Se may have a protective role in PD. 0.3 mg/kg dosage of Se increased the expression of selenoproteins, reduced the accumulation of α-Syn in the brain, and improved PD-induced motor deficits. These results suggest that Se may be a potential therapeutic option for PD treatment.

Role of cardiac ß1-adrenergic and A1-adenosine receptors in severe arrhythmias related to Parkinson's disease.

AIMS: Although reduced life expectancy in Parkinson's Disease (PD) patients has been related to severe cardiac arrhythmias due to autonomic dysfunctions, its molecular mechanisms remain unclear. To investigate the role of cardiac ß1-Adrenergic (ß1AR) and A1-Adenosine (A1R) receptors in these dysfunctions, the pharmacological effects of stimulation of cardiac ß1AR (isoproterenol, ISO), in the absence and presence of cardiac ß1AR (atenolol, AT) or A1R (1,3-dipropyl-8-cyclopentyl xanthine, DPCPX) blockade, on the arrhythmias induced by Ischemia/Reperfusion (CIR) in an animal PD model were studied. METHODS: PD was produced by dopaminergic lesions (confirmed by immunohistochemistry analysis) caused by the injection of 6-hydroxydopamine (6-OHDA, 6 µg) in rat striatum. CIR was produced by a surgical interruption for 10 min followed by reestablishment of blood circulation in the descendent left coronary artery. On the incidence of CIR-Induced Ventricular Arrhythmias (VA), Atrioventricular Block (AVB), and Lethality (LET), evaluated by Electrocardiogram (ECG) analysis, the effects of intravenous treatment with ISO, AT and DPCPX (before CIR) were studied. RESULTS: VA, AVB and LET incidences were significantly higher in 6-OHDA (83%, 92%, 100%, respectively) than in control rats (58%, 67% and 67%, respectively). ISO treatment significantly reduced these incidences in 6-OHDA (33%, 33% and 42%, respectively) and control rats (25%, 25%, 33%, respectively), indicating that stimulation of cardiac ß1AR induced cardioprotection. This response was prevented by pretreatment with AT and DPCPX, confirming the involvement of cardiac ß1AR and A1R. CONCLUSION: Pharmacological modulation of cardiac ß1AR and A1R could be a potential therapeutic strategy to reduce severe arrhythmias and increase life expectancy in PD patients.

S-adenosyl methionine improves motor co-ordination with reduced oxidative stress, dopaminergic neuronal loss, and DNA methylation in the brain striatum of 6-hydroxydopamine-induced neurodegeneration in rats.

PURPOSE: Parkinson's disease (PD) is the most common age-related neurodegenerative disease worldwide. S-adenosyl methionine (SAMe), a methyl donor that plays an important role in DNA methylation, could replenish the cellular antioxidant glutathione (GSH). Herein, we investigated the neuroprotective effects of SAMe in 6-hydroxydopamine (6-OHDA) rat models of PD and elucidated the underlying mechanism. METHODS: PD model rats were developed by injecting 6-OHDA stereotaxically into the striatum. In Phase 1 of the study, we performed the neurobehavioral tests, GSH assay, and histopathology to evaluate the neuroprotective effects of SAMe. The animals were treated with SAMe (150 or 300 mg/kg body weight) orally for 28 days. The positive control group received selegiline (5 mg/kg), whereas the disease control group received normal saline. In Phase 2, we evaluated the striatal dopamine levels and performed DNA methylation assay to uncover the mechanism of action of SAMe. In this phase, a higher dose of SAMe (300 mg/kg) was used. RESULTS: SAMe (300 mg/kg) treatment for 4 weeks significantly attenuated the abnormal circling behavior in PD rats (p < 0.05). Moreover, SAMe at both doses (150 and 300 mg/kg) enhanced the performance of PD rats in the open field test and stepping test (p < 0.05). SAMe treatment significantly increased the GSH levels, and at high dose, SAMe restricted neuronal loss in the striatum of PD-model rats (p < 0.05). Moreover, SAMe treatment led to a significant recovery in the dopamine levels and improved the DNA methylation status in the dopaminergic neurons (p < 0.05) of PD model rats. CONCLUSION: SAMe exhibits antioxidant activity and DNA methylation modulating effects in 6-OHDA model PD rats. Moreover, SAMe prevents neuronal loss in PD rats suggesting that SAMe has therapeutic potential in preventing PD development. The neuroprotective potential of SAMe is greater at high doses.

Continuous SGB Inhibits Occurrence and Maintenance of Mechanical Hyperalgesia via Reducing Inflammatory Cytokines in Striatum and PAG of PD Nociception Rat Models.

AIM: To investigate the effect of stellate ganglion block (SGB) on nociception in Parkinson's disease (PD) rat models, and to clarify the associated mechanism. MATERIAL AND METHODS: To generate PD nociception rat model 6-hydroxydopamine (6-OHDA) injection method was used. Paw withdrawal threshold (PWT) and paw retraction latency (PWL) was used to reflect mechanical stimulation and thermal stimulation, respectively, at pre-modeling and 1, 2, 3, 4 weeks post modeling. The preventive and therapeutic effects of SGB treatment on nociception were observed in Naive, Vehicle, and 6-OHDA group (model). Levels of IL-1ß, IL-6, and TNF-α in striatum and periaqueductal gray (PAG) were detected with ELISA. RESULTS: 6-OHDA injection induced obvious reduction of bilateral PWT from 2 to 4 weeks post modeling, suggesting that PD nociception rat model was successfully established. Continuous SGB prevention inhibited mechanical hyperalgesia at 2, 3 and 4 weeks post modeling, and significantly reversed mechanical hyperalgesia at 3 and 4 weeks post modeling, compared with those of Saline group (p < 0.05). These results suggest that continuous SGB could effectively prevent and alleviate pain of PD rats. SGB treatment remarkably suppressed levels of inflammatory factors (IL-1 ß, IL-6, and TNF-α) in striatum and PAG of PD rats compared with those of rats in Vehicle group (p < 0.05). CONCLUSION: Continuous SGB effectively inhibited and reversed mechanical hyperalgesia of PD nociception rats through inhibiting inflammatory response in striatum and PAG.

Human gingival mesenchymal stem cells improve movement disorders and tyrosine hydroxylase neuronal damage in Parkinson disease rats.

BACKGROUND AIMS: Gingival mesenchymal stem cells (GMSCs) demonstrate high proliferation, trilineage differentiation and immunomodulatory properties. Parkinson disease (PD) is the second most common type of neurodegenerative disease. This study aimed to explore the effect and mechanism of GMSC-based therapy in 6-hydroxydopamine-induced PD rats. METHODS: RNA sequencing and quantitative proteomics technology was used to validate the neuroprotective role of GMSCs therapeutic in 6-Hydroxydopamine -induced PD model in vitro and in vivo. Western blotting, immunofluorescence and real-time quantitative PCR verified the molecular mechanism of GMSCs treatment. RESULTS: Intravenous injection of GMSCs improved rotation and forelimb misalignment behavior, enhanced the anti-apoptotic B-cell lymphoma 2/B-cell lymphoma 2-associated X axis, protected tyrosine hydroxylase neurons, decreased the activation of astrocytes and reduced the astrocyte marker glial fibrillary acidic protein and microglia marker ionized calcium-binding adaptor molecule 1 in the substantia nigra and striatum of PD rats. The authors found that GMSCs upregulated nerve regeneration-related molecules and inhibited metabolic disorders and the activation of signal transducer and activator of transcription 3. GMSCs showed a strong ability to protect neurons and reduce mitochondrial membrane potential damage and reactive oxygen species accumulation. The safety of GMSC transplantation was confirmed by the lack of tumor formation following subcutaneous transplantation into nude mice for up to 8 weeks. CONCLUSIONS: The authors' research helps to explain the mechanism of GMSC-based therapeutic strategies and promote potential clinical application in Parkinson disease.

Human Embryonic Stem Cell-Derived Dopaminergic Grafts Alleviate L-DOPA Induced Dyskinesia.

BACKGROUND: First-in-human studies to test the efficacy and safety of human embryonic stem cells (hESC)-derived dopaminergic cells in the treatment of Parkinson's disease (PD) are imminent. Pre-clinical studies using hESC-derived dopamine neuron transplants in rat models have indicated that the benefits parallel those shown with fetal tissue but have thus far failed to consider how ongoing L-DOPA administration might impact on the graft. OBJECTIVE: To determine whether L-DOPA impacts on survival and functional recovery following grafting of hESC-derived dopaminergic neurons. METHODS: Unilateral 6-OHDA lesioned rats were administered with either saline or L-DOPA prior to, and for 18 weeks following surgical implantation of dopaminergic neural progenitors derived from RC17 hESCs according to two distinct protocols in independent laboratories. RESULTS: Grafts from both protocols elicited reduction in amphetamine-induced rotations. Reduced L-DOPA-induced dyskinesia preceded the improvement in amphetamine-induced rotations. Furthermore, L-DOPA had no effect on overall survival (HuNu) or dopaminergic neuron content of the graft (TH positive cells) but did lead to an increase in the number of GIRK2 positive neurons. CONCLUSION: Critically, we found that L-DOPA was not detrimental to graft function, potentially enhancing graft maturation and promoting an A9 phenotype. Early improvement of L-DOPA-induced dyskinesia suggests that grafts may support the handling of exogenously supplied dopamine earlier than improvements in amphetamine-induced behaviours indicate. Given that one of the protocols will be employed in the production of cells for the European STEM-PD clinical trial, this is vital information for the management of patients and achieving optimal outcomes following transplantation of hESC-derived grafts for PD.

Autonomic Regulation of Nociceptive and Immunologic Changes in a Mouse Model of Complex Regional Pain Syndrome.

Chronic pain frequently develops after limb injuries, and its pathogenesis is poorly understood. We explored the hypothesis that the autonomic nervous system regulates adaptive immune system activation and nociceptive sensitization in a mouse model of chronic post-traumatic pain with features of complex regional pain syndrome (CRPS). In studies sympathetic signaling was reduced using 6-hydroxydopamine (6-OHDA) or lofexidine, while parasympathetic signaling was augmented by nicotine administration. Hindpaw allodynia, unweighting, skin temperature, and edema were measured at 3 and 7 weeks after fracture. Hypertrophy of regional lymph nodes and IgM deposition in the skin of injured limbs were followed as indices of adaptive immune system activation. Passive transfer of serum from fracture mice to recipient B cell deficient (muMT) mice was used to assess the formation of pain-related autoantibodies. We observed that 6-OHDA or lofexidine reduced fracture-induced hindpaw nociceptive sensitization and unweighting. Nicotine had similar effects. These treatments also prevented IgM deposition, hypertrophy of popliteal lymph nodes, and the development of pronociceptive serum transfer effects. We conclude that inhibiting sympathetic or augmenting parasympathetic signaling inhibits pro-nociceptive immunological changes accompanying limb fracture. These translational results support the use of similar approaches in trials potentially alleviating persistent post-traumatic pain and, possibly, CRPS. PERSPECTIVE: Selective treatments aimed at autonomic nervous system modulation reduce fracture-related nociceptive and functional sequelae. The same treatment strategies limit pain-supporting immune system activation and the production of pro-nociceptive antibodies. Thus, the therapeutic regulation of autonomic activity after limb injury may reduce the incidence of chronic pain.

The Preventive Effect of Cardiac Sympathetic Denervation Induced by 6-OHDA on Myocardial Ischemia-Reperfusion Injury: The Changes of lncRNA/circRNAs-miRNA-mRNA Network of the Upper Thoracic Spinal Cord in Rats.

In this study, we investigated whether chemical 6-hydroxydopamine (6-OHDA) stimuli caused cardiac sympathetic denervation (SD), and we analyzed gene expression profiles to determine the changes in the lncRNA/circRNAs-miRNA-mRNA network in the affected spinal cord segments to identify putative target genes and molecular pathways in rats with myocardial ischemia-reperfusion injury (MIRI). Our results showed that cardiac sympathetic denervation induced by 6-OHDA alleviated MIRI. Compared with the ischemia reperfusion (IR, MIRI model) group, there were 148 upregulated and 51 downregulated mRNAs, 165 upregulated and 168 downregulated lncRNAs, 70 upregulated and 52 downregulated circRNAs, and 12 upregulated and 11 downregulated miRNAs in the upper thoracic spinal cord of the SD-IR group. Furthermore, we found that the differential genes related to cellular components were mainly enriched in extracellular and cortical cytoskeleton, and molecular functions were mainly enriched in chemokine activity. Pathway analysis showed that the differentially expressed genes were mainly related to the interaction of cytokines and cytokine receptors, sodium ion reabsorption, cysteine and methionine metabolism, mucoglycan biosynthesis, cGMP-PKG signaling pathway, and MAPK signaling pathway. In conclusion, the lncRNA/circRNAs-miRNA-mRNA networks in the upper thoracic spinal cord play an important role in the preventive effect of cardiac sympathetic denervation induced by 6-OHDA on MIRI, which offers new insights into the pathogenesis of MIRI and provides new targets for MIRI.

Nitroxide Radical-Containing Redox Nanoparticles Protect Neuroblastoma SH-SY5Y Cells against 6-Hydroxydopamine Toxicity.

Parkinson's disease (PD) patients can benefit from antioxidant supplementation, and new efficient antioxidants are needed. The aim of this study was to evaluate the protective effect of selected nitroxide-containing redox nanoparticles (NRNPs) in a cellular model of PD. Antioxidant properties of NRNPs were studied in cell-free systems by protection of dihydrorhodamine 123 against oxidation by 3-morpholino-sydnonimine and protection of fluorescein against bleaching by 2,2-azobis(2-amidinopropane) hydrochloride and sodium hypochlorite. Model blood-brain barrier penetration was studied using hCMEC/D3 cells. Human neuroblastoma SH-SY5Y cells, exposed to 6-hydroxydopamine (6-OHDA), were used as an in vitro model of PD. Cells were preexposed to NRNPs or free nitroxides (TEMPO or 4-amino-TEMPO) for 2 h and treated with 6-OHDA for 1 h and 24 h. The reactive oxygen species (ROS) level was estimated with dihydroethidine 123 and Fluorimetric Mitochondrial Superoxide Activity Assay Kit. Glutathione level (GSH) was measured with ortho-phtalaldehyde, ATP by luminometry, changes in mitochondrial membrane potential with JC-1, and mitochondrial mass with 10-Nonyl-Acridine Orange. NRNP1, TEMPO, and 4-amino-TEMPO (25-150 µM) protected SH-SY5Y cells from 6-OHDA-induced viability loss; the protection was much higher for NRNP1 than for free nitroxides. NRNP1 were better antioxidants in vitro and permeated better the model BBB than free nitroxides. Exposure to 6-OHDA decreased the GSH level after 1 h and increased it considerably after 24 h (apparently a compensatory overresponse); NRNPs and free nitroxides prevented this increase. NRNP1 and free nitroxides prevented the decrease in ATP level after 1 h and increased it after 24 h. 6-OHDA increased the intracellular ROS level and mitochondrial superoxide level. Studied antioxidants mostly decreased ROS and superoxide levels. 6-OHDA decreased the mitochondrial potential and mitochondrial mass; both effects were prevented by NRNP1 and nitroxides. These results suggest that the mitochondria are the main site of 6-OHDA-induced cellular damage and demonstrate a protective effect of NRNP1 in a cellular model of PD.

Protective role of microRNA-221 in Parkinson's disease.

OBJECTIVE: The present study aimed to explore the role and the underlying mechanism of miR-221 in Parkinson's Disease. MATERIALS AND METHODS: To perform our investigation, a PD cell model was created by using 6-OHDA. Cell viability and proliferation assays, and flow cytometry analysis were performed to detect cell viability and apoptosis. The qRT-PCR and western blotting were used for gene and protein level detection. RESULTS: We found that the expression of miRNA-221 is significantly lower in 6-OHDA treated PC12 pheochromocytoma cells compared to the normal cells. The results of further analysis indicated that miR-221 mimic significantly promoted the cell viability and proliferation of PC12 cells treated with 6-OHDA. MiR-221 mimic significantly inhibited 6-OHDA-treated PC12 cells from apoptosis. These effects were eliminated by PTEN over-expression. We also revealed that PTEN was a direct target gene of miR-221. Moreover, we found miR-221 mimic significantly promoted the phosphorylation of AKT in PC12 cells treated with 6-OHDA, and over-expression of PTEN could eliminate this effect. CONCLUSIONS: MiR-221 plays a protective role in Parkinson's Disease via regulating PC12 cell viability and apoptosis by targeting PTEN. Therefore, miR-221 may serve as a potential therapeutic target for Parkinson's disease treatment (Fig. 3, Ref. 27).

Restoring Spinal Noradrenergic Inhibitory Tone Attenuates Pain Hypersensitivity in a Rat Model of Parkinson's Disease.

In the present study, we investigated whether restoring descending noradrenergic inhibitory tone can attenuate pain in a PD rat model, which was established by stereotaxic infusion of 6-hydroxydopamine (6-OHDA) into the bilateral striatum (CPu). PD rats developed thermal and mechanical hypersensitivity at the 4th week after surgery. HPLC analysis showed that NE content, but not dopamine or 5-HT, significantly decreased in lumbar spinal cord in PD rats. Additional noradrenergic depletion by injection of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) aggravated pain hypersensitivity in PD rats. At the 5th week after injection of 6-OHDA, systemic treatment with pharmacological norepinephrine (NE) precursor droxidopa (L-DOPS) or α2 adrenoceptor agonist clonidine significantly attenuated thermal and mechanical pain hypersensitivity in PD rats. Furthermore, application of norepinephrine (NE) and 5-hydroxytryptamine (5-HT) reuptake inhibitors duloxetine, but not 5-HT selective reuptake inhibitors sertraline, significantly inhibited thermal and mechanical pain hypersensitivity in PD rats. Systemic administration of Madopar (L-DOPA) or the D2/D3 agonist pramipexole slightly inhibited the thermal, but not mechanical, hypersensitivity in PD rats. Thus, our study revealed that impairment of descending noradrenergic system may play a key role in PD-associated pain and restoring spinal noradrenergic inhibitory tone may serve as a novel strategy to manage PD-associated pain.

Neuroprotective effect of combined therapy with hyperbaric oxygen and madopar on 6-hydroxydopamine-induced Parkinson's disease in rats.

Parkinson's disease (PD) is a common movement disorder in the elderly. In the present study, we examined whether the combination of hyperbaric oxygen (HBO) and madopar therapy provided a neuroprotective effect on dopaminergic neurons in the substantia nigra using a rat model of PD. Rotational assessments revealed that both HBO and combination therapy significantly attenuated apomorphine-induced turning in PD rats. Our results indicated that the combination therapy increased glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities and reduced the malondialdehyde (MDA) content in the SN. Furthermore, the combination therapy resulted in significant protection against the loss of neurons, and specifically tyrosine hydroxylase (TH)-positive neurons, in the SN and also alleviated the production of glial fibrillary acidic protein (GFAP). The levels of Bcl-2 were increased and Bax were decreased following the HBO or combination therapy. In brief, the neuroprotective effect of combined therapy with HBO and madopar against 6-OHDA-induced PD rats may rely on its ability to reduce oxidative stress and protect against Bax/Bcl-2-mediated apoptosis.

Trans-blood brain barrier delivery of dopamine-loaded nanoparticles reverses functional deficits in parkinsonian rats.

Sustained and safe delivery of dopamine across the blood brain barrier (BBB) is a major hurdle for successful therapy in Parkinson's disease (PD), a neurodegenerative disorder. Therefore, in the present study we designed neurotransmitter dopamine-loaded PLGA nanoparticles (DA NPs) to deliver dopamine to the brain. These nanoparticles slowly and constantly released dopamine, showed reduced clearance of dopamine in plasma, reduced quinone adduct formation, and decreased dopamine autoxidation. DA NPs were internalized in dopaminergic SH-SY5Y cells and dopaminergic neurons in the substantia nigra and striatum, regions affected in PD. Treatment with DA NPs did not cause reduction in cell viability and morphological deterioration in SH-SY5Y, as compared to bulk dopamine-treated cells, which showed reduced viability. Herein, we report that these NPs were able to cross the BBB and capillary endothelium in the striatum and substantia nigra in a 6-hydroxydopamine (6-OHDA)-induced rat model of PD. Systemic intravenous administration of DA NPs caused significantly increased levels of dopamine and its metabolites and reduced dopamine-D2 receptor supersensitivity in the striatum of parkinsonian rats. Further, DA NPs significantly recovered neurobehavioral abnormalities in 6-OHDA-induced parkinsonian rats. Dopamine delivered through NPs did not cause additional generation of ROS, dopaminergic neuron degeneration, and ultrastructural changes in the striatum and substantia nigra as compared to 6-OHDA-lesioned rats. Interestingly, dopamine delivery through nanoformulation neither caused alterations in the heart rate and blood pressure nor showed any abrupt pathological change in the brain and other peripheral organs. These results suggest that NPs delivered dopamine into the brain, reduced dopamine autoxidation-mediated toxicity, and ultimately reversed neurochemical and neurobehavioral deficits in parkinsonian rats.

Magnetic resonance imaging (MRI) to study striatal iron accumulation in a rat model of Parkinson's disease.

Abnormal accumulation of iron is observed in neurodegenerative disorders. In Parkinson's disease, an excess of iron has been demonstrated in different structures of the basal ganglia and is suggested to be involved in the pathogenesis of the disease. Using the 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease, the edematous effect of 6-OHDA and its relation with striatal iron accumulation was examined utilizing in vivo magnetic resonance imaging (MRI). The results revealed that in comparison with control animals, injection of 6-OHDA into the rat striatum provoked an edematous process, visible in T2-weighted images that was accompanied by an accumulation of iron clearly detectable in T2*-weighted images. Furthermore, Prussian blue staining to detect iron in sectioned brains confirmed the existence of accumulated iron in the areas of T2* hypointensities. The presence of ED1-positive microglia in the lesioned striatum overlapped with this accumulation of iron, indicating areas of toxicity and loss of dopamine nerve fibers. Correlation analyses demonstrated a direct relation between the hyperintensities caused by the edema and the hypointensities caused by the accumulation of iron.

Behavioral sensitization to different dopamine agonists in a parkinsonian rodent model of drug-induced dyskinesias.

Repeated treatment with dopamine (DA) receptor agonists strongly potentiates contralateral turning behavior due to selective stimulation of D1 or D2-class receptors in 6-hydroxydopamine (6-OHDA)-lesioned rats. This phenomenon, referred to as sensitization, is believed to be related to the motor response complications (dyskinesias, on-off states) that occur during chronic administration of levodopa in Parkinson's disease patients. In recent years a new method for the evaluation of abnormal involuntary movements (AIMs) secondary to dopaminergic stimulation in 6-OHDA-lesioned rats was described. These AIMs resemble dyskinesias as seen in parkinsonian patients under levodopa therapy. Our objective was to evaluate the effects of repeated treatment with different regimes of DA agonists on turning behavior and on an AIMs scale in 6-OHDA lesioned rats, with the aim of discriminating between drugs with different dyskinesia-inducing potential. In addition, we explored the effects of a previous exposure to a DA agonist (priming) on the behavioral response to the subsequent administration of a DA agonist with the same or different pharmacologic profile. Our results show that in apomorphine-treated rats, rotational behavior and AIMs run a parallel course of enhancement, while in those receiving quinpirole there is a dissociation, suggesting that they could be mediated by different mechanisms. The finding of a significant priming effect on subsequent testing of 6-OHDA lesioned rats should be borne in mind as the use of these pharmacological tests in the screening of well lesioned animals could lead to an erroneous interpretation of further results on dyskinesias and rotational behavior.

Adjunctive treatment of murine neuroblastoma with 6-hydroxydopamine and Tempol.

Currently available therapy for disseminated neuroblastoma affords only a 5-20% 5-year survival rate. We have attempted to design targeted chemotherapy for this disease by exploiting the dopamine uptake system on neuroblastoma cells. 6-Hydroxydopamine (6OHDA) is a neurotransmitter analogue, which generates cytolytic oxygen radicals in neuroblastoma cells that take it up. It is, however, predictably, systemically toxic, because of its spontaneous oxidation. Its toxicity is particularly severe in the sympathetic nervous system, because this tissue selectively concentrates dopamine and its analogues. Lowering the dose of 6OHDA below toxic levels prohibitively compromises its antitumor effect. To avoid both the systemic and sympathetic nervous system toxicity yet retain the antitumor efficacy of 6OHDA, we have used the antioxidant Tempol adjunctively with 6OHDA. Administration of Tempol (250 mg/kg, i.p.) 10 min prior to administration of toxic doses of 6OHDA (350 or 400 mg/kg, i.p.) resulted in a decrease in the mortality rate, sympathetic nervous system impairment, and activity impairment compared with those seen with 6OHDA alone. Tumor weights from mice administered saline or Tempol alone were 3.6 +/- 1.9 and 2.9 +/- 0.7 g, respectively. In contrast, mice administered Tempol followed by 6OHDA had an average tumor weight of 0.7 +/- 0.3 g. Tumor incidence was also reduced from 80-100% to 40%. Studies performed using electron spin resonance spectroscopy suggest that Tempol acts in this system by reacting directly with both the 6OHDA radical and, in the presence of iron, its oxidation product, the hydroxyl radical.

Inhibitions by 6-hydroxydopamine and neostigmine singly or together of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.

The effects of chemical sympathectomy induced by 6-hydroxydopamine (6-OHDA) and administration of the acetylcholinesterase inhibitor neostigmine, singly or together, on gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and on the tissue catecholamine concentration of the gastric wall and the labeling index of the gastric mucosa, were investigated in inbred Wistar rats. Rats received s.c. injections of neostigmine (0.075 mg/kg), and/or i.p. injections of 6-OHDA (42 mg/kg twice within 24 hr, and then 105 mg/kg every 2 weeks from 1 week later) 25 weeks after oral treatment with MNNG. Prolonged administration of 6-OHDA or neostigmine significantly reduced the incidence of gastric cancers by week 52, and in combination they had a significantly greater inhibitory effect. 6-OHDA and/or neostigmine had no influence on the histology of gastric cancers. Administration of 6-OHDA, but not neostigmine, significantly decreased the norepinephrine concentration in the antral portion of the gastric wall. The labeling index of the antral mucosa was decreased significantly by treatment with 6-OHDA or neostigmine, and decreased even more significantly by 6-OHDA plus neostigmine. Our findings indicate that 6-OHDA and neostigmine have protective effects against gastric carcinogenesis and that in combination their effects are additive. These results imply that the activities of the sympathetic and parasympathetic autonomic systems together influence gastric carcinogenesis.

A neurologist's approach to neuroblastoma.

Neuroblastoma is among the most common malignancies of childhood. Despite greatly improved therapy for some pediatric tumors, the prognosis for children with metastatic neuroblastoma has not changed significantly in the past 10 years. With conventional chemotherapy, radiation therapy, and surgery, children with metastatic neuroblastoma have a 20% long-term survival rate. We have pursued novel chemotherapeutic approaches to neuroblastoma that target the neurotransmitter receptors on the surface of these cells. Specificity for these neural crest tumor cells is effected by (1) selective protection of normal neuronal elements from toxicity, or (2) selective potentiation of toxicity for neural tumor cells. In the first instance, the oxygen radical-generating neurotransmitter analogue 6-hydroxydopamine is used as a neural crest-specific toxin. Normal neural crest cells are protected from this toxicity by oxygen radical-scavenging analogues of the compound WR2721, which is specifically taken up by nonneoplastic cells. In the second instance, neocarzinostatin, an antineoplastic natural product that must be activated by thiol groups to be toxic, is used in conjunction with 6-mercaptodopamine, a thiol-containing compound that gains specific entry into neural crest cells by virtue of its neurotransmitter-like structure. We have found that neocarzinostatin induces morphologic differentiation of neuroblastoma cells, and we are also currently characterizing the biochemical accompaniments of this morphologic change.

Inhibition of azoxymethane-induced experimental colon carcinogenesis in Wistar rats by 6-hydroxydopamine.

The effects of 6-hydroxydopamine (6-OHDA) on the incidence, number and histology of colon tumors induced by azoxymethane (AOM), and on norepinephrine (NE) concentration in the colon wall and the labelling index of the colon mucosa were investigated in Wistar rats. Rats received sub-cutaneous (s.c.) injections of AOM once a week for 10 weeks, and throughout 35 weeks were also given intraperitoneal (i.p.) injections of 6-OHDA. Prolonged administration of 6-OHDA was found to cause a significant reduction in the incidence and number of colon tumors. However, it had no influence on the histological features or depths of involvement of colon tumors and/or adenocarcinomas. Its administration also caused significant decreases in the NE concentration in the colon wall and in the labelling index of the colon mucosa. Our findings indicate that 6-OHDA has a protective effect against colon carcinogenesis, and that the activity of the sympathetic nervous system may have an important influence on colon carcinogenesis.