Mariza Bortolanza

Glial activation is associated with l-DOPA induced dyskinesia and blocked by a nitric oxide synthase inhibitor in a rat model of Parkinson's disease.

l-3, 4-dihydroxyphenylalanine (L-DOPA) is the most effective treatment for Parkinsons disease but can induce debilitating abnormal involuntary movements (dyskinesia). Here we show that the development of L-DOPA-induced dyskinesia in the rat is accompanied by upregulation of an inflammatory cascade involving nitric oxide. Male Wistar rats sustained unilateral injections of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle. After three weeks animals started to receive daily treatment with L-DOPA (30 mg/kg plus benserazide 7.5 mg/kg, for 21 days), combined with an inhibitor of neuronal NOS (7-nitroindazole, 7-NI, 30 mg/kg/day) or vehicle (saline-PEG 50%). All animals treated with L-DOPA and vehicle developed abnormal involuntary movements, and this effect was prevented by 7-NI. L-DOPA-treated dyskinetic animals exhibited an increased striatal and pallidal expression of glial fibrillary acidic protein (GFAP) in reactive astrocytes, an increased number of CD11b-positive microglial cells with activated morphology, and the rise of cells positive for inducible nitric oxide-synthase immunoreactivity (iNOS). All these indexes of glial activation were prevented by 7-NI co-administration. These findings provide evidence that the development of L-DOPA-induced dyskinesia in the rat is associated with activation of glial cells that promote inflammatory responses. The dramatic effect of 7-NI in preventing this glial response points to an involvement of nitric oxide. Moreover, the results suggest that the NOS inhibitor prevents dyskinesia at least in part via inhibition of glial cell activation and iNOS expression. Our observations indicate nitric oxide synthase inhibitors as a therapeutic strategy for preventing neuroinflammatory and glial components of dyskinesia pathogenesis in Parkinsons disease.

Are cyclooxygenase-2 and nitric oxide involved in the dyskinesia of Parkinson's disease induced by l-DOPA?

Inflammatory mechanisms are proposed to play a role in l-DOPA-induced dyskinesia. Cyclooxygenase-2 (COX2) contributes to inflammation pathways in the periphery and is constitutively expressed in the central nervous system. Considering that inhibition of nitric oxide (NO) formation attenuates l-DOPA-induced dyskinesia, this study aimed at investigating if a NO synthase (NOS) inhibitor would change COX2 brain expression in animals with l-DOPA-induced dyskinesia. To this aim, male Wistar rats received unilateral 6-hydroxydopamine microinjection into the medial forebrain bundle were treated daily with l-DOPA (21 days) combined with 7-nitroindazole or vehicle. All hemi-Parkinsonian rats receiving l-DOPA showed dyskinesia. They also presented increased neuronal COX2 immunoreactivity in the dopamine-depleted dorsal striatum that was directly correlated with dyskinesia severity. Striatal COX2 co-localized with choline-acetyltransferase, calbindin and DARPP-32 (dopamine-cAMP-regulated phosphoprotein-32), neuronal markers of GABAergic neurons. NOS inhibition prevented l-DOPA-induced dyskinesia and COX2 increased expression in the dorsal striatum. These results suggest that increased COX2 expression after l-DOPA long-term treatment in Parkinsonian-like rats could contribute to the development of dyskinesia.

l-DOPA-induced dyskinesia and neuroinflammation: do microglia and astrocytes play a role?

In Parkinsons disease (PD), l‐DOPA therapy leads to the emergence of motor complications including l‐DOPA‐induced dyskinesia (LID). LID relies on a sequence of pre‐ and postsynaptic neuronal events, leading to abnormal corticostriatal neurotransmission and maladaptive changes in striatal projection neurons. In recent years, additional non‐neuronal mechanisms have been proposed to contribute to LID. Among these mechanisms, considerable attention has been focused on l‐DOPA‐induced inflammatory responses. Microglia and astrocytes are the main actors in neuroinflammatory responses, and their double role at the interface between immune and neurophysiological responses is starting to be elucidated. Both microglia and astrocytes express a multitude of neurotransmitter receptors and via the release of several soluble molecules modulate synaptic function in neuronal networks. Here we review preclinical and clinical evidence of glial overactivation by l‐DOPA, supporting a role of microglia and astrocytes in the development of LID. We propose that in PD, chronically and abnormally activated microglia and astrocytes lead to an aberrant neuron‐glia communication, which affect synaptic activity and neuroplasticity contributing to the development of LID.

l‐DOPA‐induced dyskinesia in Parkinson's disease: Are neuroinflammation and astrocytes key elements?

Inflammation in Parkinsons disease (PD) is a new concept that has gained ground due to the potential of mitigating dopaminergic neuron death by decreasing inflammation. The solution to this question is likely to be complex. We propose here that the significance of inflammation in PD may go beyond the nigral cell death. The pathological process that underlies PD requires years to reach its full extent. A growing body of evidence has been accumulated on the presence of multiple inflammatory signs in the brain of PD patients even in very late stages of the disease. Because neuron‐microglia‐astrocyte interactions play a major role in the plasticity of neuronal response to l‐DOPA in post‐synaptic neurons, we focused this review on our recent results of l‐DOPA‐induced dyskinesia in rodents correlating it to significant findings regarding glial cells and neuroinflammation. We showed that in the rat model of PD/l‐DOPA‐induced dyskinesia there was an increased expression of inflammatory markers, such as the enzymes COX2 in neurons and iNOS in glial cells, in the dopamine‐denervated striatum. The gliosis commonly seem in PD was associated with modifications in astrocytes and microglia that occur after chronic treatment with l‐DOPA. Either as a cause, consequence, or promoter of progression of neuronal degeneration, inflammation plays a role in PD. The key aims of current PD research ought to be to elucidate (a) the time sequence in which the inflammatory factors act in PD patient brain and (b) the mechanisms by which neuroinflammatory response contributes to the collateral effects of l‐DOPA treatment.

Neuroprotective effect of omega-3 polyunsaturated fatty acids in the 6-OHDA model of Parkinson's disease is mediated by a reduction of inducible nitric oxide synthase

Objective: Parkinsons disease (PD) is characterized by deterioration of the nigrostriatal system and associated with chronic neuroinflammation. Glial activation has been associated with regulating the survival of dopaminergic neurons and is thought to contribute to PD through the release of proinflammatory and neurotoxic factors, such as reactive nitric oxide (NO) that triggers or exacerbates neurodegeneration in PD. Polyunsaturated fatty acids (PUFAs) exert protective effects, including antiinflammatory, antiapoptotic, and antioxidant activity, and may be promising for delaying or preventing PD by attenuating neuroinflammation and preserving dopaminergic neurons. The present study investigated the effects of fish oil supplementation that was rich in PUFAs on dopaminergic neuron loss, the density of inducible nitric oxide synthase (iNOS)-immunoreactive cells, and microglia and astrocyte reactivity in the substantia nigra pars compacta (SNpc) and striatal dopaminergic fibers. Methods: The animals were supplemented with fish oil for 50 days and subjected to unilateral intrastriatal 6-hydroxydopamine (6-OHDA)-induced lesions as a model of PD. Results: Fish oil mitigated the loss of SNpc neurons and nerve terminals in the striatum that was caused by 6-OHDA. This protective effect was associated with reductions of the density of iNOS-immunoreactive cells and microglia and astrocyte reactivity. Discussion: These results suggest that the antioxidant and antiinflammatory properties of fish oil supplementation are closely related to a decrease in dopaminergic damage that is caused by the 6-OHDA model of PD. GRAPHICAL ABSTRACT

Fish oil prevents rodent anxious states comorbid with diabetes: A putative involvement of nitric oxide modulation

HighlightsDiabetic rats exhibited a pronounced anxiogenic‐like behavior.Fish oil treatment prevented the anxiogenic‐like behavior in diabetic animals.Fish oil treatment prevented the high nNOS expression in brain areas related to anxiety.Treatment with nitric oxide precursor abolished the anxiolytic effect induced by fish oil. Abstract There is an urgent need to understand the pathophysiological mechanisms related to anxiety associated with diabetes, seeking more effective alternative treatments to treat it. For that, the effect of a preventive and prolonged treatment with fish oil (FO), a source of omega‐3 polyunsaturated fatty acid, was tested in streptozotocin‐diabetic (DBT) rats submitted to the anxiety tests. Additionally, an immunohistochemistry for neuronal NO synthase (nNOS) was performed in brain areas related to anxiety, such as lateral amygdala (AMY), hippocampus (HIP) and dorsolateral periaqueductal gray (dlPAG). Lastly, the effect of NO precursor L‐arginine (L‐Arg) or nNOS inhibitor 7‐nitroindazole (7‐NI) was tested in DBT animals treated with vehicle (VEH) or FO. Our data demonstrated that vehicle‐treated DBT animals exhibited a more pronounced anxiogenic‐like response and also presented high nNOS levels in the AMY, HIP and rostral dlPAG, what were both significantly prevented by FO treatment. This treatment was able to prevent the impairment in locomotor activity besides improving the high glycemic levels in DBT rats. Interestingly, while injection of 7‐NI or L‐Arg in VEH‐treated DBT animals induced an anxiogenic‐like and anxiolytic‐like effect, respectively; the previous treatment with both L‐Arg and 7‐NI in FO‐DBT animals abolished the anxiolytic‐like effect induced by FO treatment. Altogether, our data support the hypothesis that a dysregulation in the NO production in brain areas as AMY, HIP and dlPAG may contribute to the mechanisms that link anxiety and diabetes, and the prevention of nNOS brain expression changes induced by a prolonged treatment with FO may be an important mechanism related to its anxiolytic‐like effect.

Nanomedicine to Overcome Current Parkinson’s Treatment Liabilities: A Systematic Review

Nanoparticles might be produced and manipulated to present a large spectrum of properties. The physicochemical features of the engineered nanomaterials confer to them different features, including the ability to cross the blood–brain barrier. The main objective of this review is to present the state-of-art research in nano manipulation concerning Parkinson’s disease (PD). In the past few years, the association of drugs with nanoparticles solidly improved treatment outcomes. We systematically reviewed 28 studies, describing their potential contributions regarding the role of nanomedicine to increase the efficacy of known pharmacological strategies for PD treatment. Data from animal models resulted in the (i) improvement of pharmacological properties, (ii) more stable drug concentrations, (iii) longer half-live and (iv) attenuation of pharmacological adverse effects. As this approach is recent, with many of the described works being published less than 5 years ago, the expectancy is that this knowledge gives support to an improvement in the current clinical methods to the management of PD and other neurodegenerative diseases.

Nociceptive Response to l -DOPA-Induced Dyskinesia in Hemiparkinsonian Rats

Non-motor symptoms are increasingly identified to present clinical and diagnostic importance for Parkinson’s disease (PD). The multifactorial origin of pain in PD makes this symptom of great complexity. The dopamine precursor, l-DOPA (l-3,4-dihydroxyphenylalanine), the classic therapy for PD, seems to be effective in pain threshold; however, there are no studies correlating l-DOPA-induced dyskinesia (LID) and nociception development in experimental Parkinsonism. Here, we first investigated nociceptive responses in a 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson’s disease to a hind paw-induced persistent inflammation. Further, the effect of l-DOPA on nociception behavior at different times of treatment was investigated. Pain threshold was determined using von Frey and Hot Plate/Tail Flick tests. Dyskinesia was measured by abnormal involuntary movements (AIMs) induced by l-DOPA administration. This data is consistent to show that 6-OHDA-lesioned rats had reduced nociceptive thresholds compared to non-lesioned rats. Additionally, when these rats were exposed to a persistent inflammatory challenge, we observed increased hypernociceptive responses, namely hyperalgesia. l-DOPA treatment alleviated pain responses on days 1 and 7 of treatment, but not on day 15. During that period, we observed an inverse relationship between LID and nociception threshold in these rats, with a high LID rate corresponding to a reduced nociception threshold. Interestingly, pain responses resulting from CFA-induced inflammation were significantly enhanced during established dyskinesia. These data suggest a pro-algesic effect of l-DOPA-induced dyskinesia, which is confirmed by the correlation founded here between AIMs and nociceptive indexes. In conclusion, our results are consistent with the notion that central dopaminergic mechanism is directly involved in nociceptive responses in Parkinsonism condition.

Repurposing an established drug: an emerging role for methylene blue in L-DOPA-induced dyskinesia

The nitric oxide (NO) system has been proven to be a valuable modulator of L‐DOPA‐induced dyskinesia in Parkinsonian rodents. NO activates the enzyme soluble guanylyl cyclase and elicits the synthesis of the second‐messenger cGMP. Although we have previously described the anti‐dyskinetic potential of NO synthase inhibitors on L‐DOPA‐induced dyskinesia, the effect of soluble guanylyl cyclase inhibitors remains to be evaluated. The aim of this study was to analyze whether the clinically available non‐selective inhibitor methylene blue, or the selective soluble guanylyl cyclase inhibitor ODQ (1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one), could mitigate L‐DOPA‐induced dyskinesia in 6‐hydroxydopamine‐lesioned rats. Here, we demonstrated that methylene blue was able to reduce L‐DOPA‐induced dyskinesia incidence when chronically co‐administered with L‐DOPA during 3 weeks. Methylene blue chronic (but not acute) administration (2 weeks) was effective in attenuating L‐DOPA‐induced dyskinesia in rats rendered dyskinetic by a previous course of L‐DOPA chronic treatment. Furthermore, discontinuous methylene blue treatment (e.g., co‐administration of methylene blue and L‐DOPA for 2 consecutive days followed by vehicle and L‐DOPA co‐administration for 5 days) was effective in attenuating dyskinesia. Finally, we demonstrated that microinjection of methylene blue or ODQ into the lateral ventricle effectively attenuated L‐DOPA‐induced dyskinesia. Taken together, these results demonstrate an important role of NO‐soluble guanylyl cyclase‐cGMP signaling on L‐DOPA‐induced dyskinesia. The clinical implications of this discovery are expected to advance the treatment options for patients with Parkinsons disease.

Differential behavioral and glial responses induced by dopaminergic mechanisms in the iNOS knockout mice

HIGHLIGHTSiNOS deficient mice shows altered basal exploratory behavior.Either haloperidol or reserpine induces different effects in iNOS deficient mice.There is decreased activation of astrocytes and microglia in iNOS deficient mice.iNOS deficient mice showed decreased astroglial reaction induced by reserpine. ABSTRACT The interaction between distinctive nitric oxide synthase (NOS) isoforms and the dopamine system provides new avenues to the development of pharmacological tools for the pathophysiological conditions of the dopaminergic system. Our aim was to investigate the influences of dopamine‐induced effects in inducible NOS knockout (iNOS KO) mice. In order to characterize iNOS KO mice phenotype, the animals were submitted to the basal analyses of motor, sensorimotor and sensorial abilities. Pharmacological challenging of the dopaminergic system included the investigation of amphetamine‐induced prepulse inhibition (PPI) disruption, haloperidol‐induced catalepsy, reserpine‐induced oral involuntary movements and hyperlocomotion induced by amphetamine in reserpine treated mice. The iNOS KO mice showed significant reduction of spontaneous motor activity, but there was no significant difference in sensorimotor or sensorial responses of iNOS KO mice compared to wild type (WT). Regarding the dopaminergic system, iNOS KO mice showed a significant increase of haloperidol‐induced catalepsy. This effect was confirmed through an iNOS pharmacological inhibitor (1400W) in WT mice. In addition, iNOS KO reserpine treated mice showed reduced oral involuntary movements and amphetamine‐induced hyperlocomotion. Knowing that iNOS is mainly expressed in glial cells we analyzed the immunoreactivity (ir) for GFAP (astrocyte marker) and IBA‐1 (microglial marker) in the striatum, an area enrolled in motor planning among other functions. iNOS KO presented reduced GFAP‐ir and IBA‐1‐ir compared with WT. Reserpine treatment increased GFAP‐ir in both WT and iNOS KO. However, these effects were slighter in iNOS KO. Activated state of microglia was increased by reserpine only in WT mice. Our results further demonstrated that the absence of iNOS interfered with dopamine‐mediated behavioral and molecular responses. These results increase the understanding of the dopamine and NO system interaction, which is useful for the management of the dopamine‐related pathologies.