MAIN POINTS
Abbreviations - 6-OHDA: 6-hydroxydopamine; AIMs: axial, limb and orofacial abnormal involuntary movements; ALO: global AIMs Score including all AIM categories: axial, forelimb, and orofacial; DOXY: doxycycline; GFAP: glial fibrillary acidic protein from astrocyte; L-DOPA: 3,4-dihydroxyphenyl-L-alanine; LID: L-DOPA-induced dyskinesia; NG2-glia: Nerve/glial antigen 2 glial cells; OX-42: CD11b/c equivalent protein of microglia; PD: Parkinson’s disease; SNc: substantia nigra compacta; TH: tyrosine hydroxylase.
INTRODUCTION (742 words)
Parkinson’s disease (PD) is a neurodegenerative disorder, triggered by the progressive loss of dopamine-producing neurons in the substantia nigra compacta (SNc) region of the basal ganglia (Vaillancourt and Lehericy, 2018). The disease was considered mainly by motor symptomatology, including resting tremor, bradykinesia, limb rigidity, and defects in gait and balance (Obeso et al., 2018). Currently, symptomatic therapies are available, mainly the dopamine replacement therapy with levodopa (L-DOPA: l-3,4-dihydroxyphenylalanine). L-DOPA improves motor symptoms, but long-term L-DOPA use leads to the gradual development of side effects such as on-off fluctuations, abnormal involuntary movements, and hallucinations (Obeso et al., 2000; Dauer and Przedborski, 2003). The abnormal involuntary movements termed L-DOPA-induced dyskinesia (LID), are among the main challenges in treating PD since they limit the L-DOPA effectiveness.
To date, LID cause is not entirely understood (Fahn et al., 2004; Olanow et al., 2004; Jenner, 2008). Several studies aimed to find alternative therapies that reduce LID (Cenci et al., 2020). It was recently demonstrated the presence of an inflammatory reaction in the brains of patients exhibiting a history of dyskinesia (Del-Bel et al., 2016; Carta et al., 2017; Junior et al., 2020). In the PD post mortem brain patients, the basal ganglia is marked by angiogenesis, vascular endothelial growth factor up-regulation, and altered brain blood barrier properties (Ohlin et al., 2011, 2012; Janelidze et al., 2015; Lerner et al., 2017). Pre-clinical studies showed evidence of an inflammatory environment in the dopamine depleted striatum, with sustained activation of astrocytes and microglia and the recruitment of immune elements contributing to the pathophysiology of LID (Picconi et al., 2002; Robelet et al., 2004; Meissner et al., 2006; Buck and Ferger, 2010; Bortolanza et al., 2015a; Del-Bel et al et al., 2016; Guerra et al., 2019). Supporting the hypothesis, the striatum of lesioned rats long after the microinjection of the neurotoxin 6-hydroxydopamine (6-OHDA), receiving L-DOPA treatment, has been revealed a sustained neuroinflammatory reaction (Spinnewyn et al., 2011; Aron-Badin et al., 2013; Muñoz et al., 2014; Bortolanza et al., 2015a and b; Ramirez-Garcia et al., 2016; Teema et al., al., 2016; Boi et al., 2019). Targeting neuroinflammation may be a strategy to limit LID (Del Bel et al., 2016; Dos-Santos-Pereira et al., 2016; Carta et al., 2017; Junior et al., 2020).
A population of dividing glial progenitors, called NG2-glia, considered the fourth glial type in the adult central nervous system, has been identified throughout adult brain parenchyma (Dimou et al. 2008; Richardson et al., 2011). This population express markers typically found in oligodendrocyte precursor cells during development such as chondroitin sulfate proteoglycan (Levine et al., 1998; Peters, 2004; Nishiyama et al., 2009). It has been reported their potential to generate a wide variety of cell types besides oligodendrocytes, including astrocytes and neurons (Belachew et al., 2003; Aguirre and Gallo, 2004; Aguirre et al., 2010; Baracskay et al., 2007), although the latter is debated (Dimou et al., 2008; Nishiyama et al., 2009; Zhu et al., 2011; Richardson et al., 2011). Rodent NG2 glia receives direct synaptic inputs from glutamatergic and GABAergic neurons, a feature that is unique among glial cells (Bergles, et al., 2000; Lin & Bergles, 2004). Furthest, it was demonstrated that neuronal activity promotes the recruitment and differentiation of NG2 cells in the adult brain, which could contribute to neural plasticity (Gibson et al., 2014).
NG2-glia reacts to many types of injury or pathological conditions changing their morphology and proliferation rate. Additionally, NG2-glia responds to inflammatory cues, exhibiting a behaviour remarkably similar to microglial cells (Kang et al., 2013; Wang et al., 2017). NG2-glia surveys their microenvironment through constant filopodia extension changing their morphology (Nishiyama et al., 1997; Martín-López et al., 2013; Bribian et al., 2018; Okada et al., 2018). Zhang et al. (2019) described a downregulation of NG2-glia expression in the SNc of PD patient brain compared with healthy subjects. Kitamura et al. (2010) detected activated NG2-positive cells in the nigral 6-OHDA-injected model but not in the striatum. These findings suggest the dysfunction of NG2 glia in the PD brain and provide a compelling rationale for developing new studies.
Here we analyzed the NG2-glia response in the striatum of parkinsonian rats expressing LID using immunoconfocal morphometry, immunohistochemistry and immunoblotting. We examined NG2-glia distribution in the lesioned striatum, the cells’ phenotypic characteristics, and the association with astrocytes and microglia cells. Because molecules capable of modulating glial cells’ activation were effective in resolving LID, we determine the antidyskinetic effect of doxycycline (6-Deoxy-5-hydroxytetracycline, Bortolanza et al., 2020), in NG2 expression and NG2-glia activation.
METHODS (2.243 words)
Subjects Adult, male Wistar rats (n=64, 250–300 g, aged 9–11 weeks) were used in this study. Animals were housed in groups of three per cage, maintained at a temperature of 22–25 °C, on a 12-h light/dark cycle, and with food and water (autoclaved tap water) available ad libitum. Studies regarding sex differences, which may produce biological variables, were not investigated in this study. The experiments were performed in compliance with the recommendations of the US National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Sao Paulo (Approval Number: 2017-0014-02). All efforts of the researchers minimized the animal suffering and the number of animals used.
Drugs The dose regimen and route of administration of drugs were based on previously published studies (Cenci et al., 1998; Gomes et al., 2008; Lazzarini et al., 2013; Padovan-Neto et al., 2015). L-DOPA (L-3,4-Dihydroxyphenylalanine methyl ester hydrochloride; 20 mg/kg orally- Prolopa dispersive, Hoffman-LaRoche, Rio de Janeiro, RJ, Brazil), plus benserazide–HCl (5 mg/kg) were dissolved in water. Doxycycline (40 mg/kg i. p., Sigma-Aldrich, St. Louis, MO, USA) was dissolved in saline and administered 30 min before L-DOPA. All drugs and their respective vehicle (VEH) were freshly prepared before use and injected in a volume of 1mL/kg.
Parkinsonian Lesion Microinjection of 6-OHDA was delivered into the medial forebrain bundle as previously described (Gomes and Del-Bel, 2003; Gomes et al., 2008; Padovan-Neto et al., 2009). Animals were anaesthetized with 2,2,2-tribromoethanol (Sigma-Aldrich, St. Louis, MO, USA‎) (250 mg kg-1, i.p.) and fixed into the stereotaxic apparatus for performing the surgery (David Kopf, model USA, 9:57). Stereotaxic coordinates were (from bregma in mm: AP = -4.3; LL = -1.6; DV = -8.3) were based on Paxinos & Watson, (2007). Rats received microinjection of 6-OHDA in a volume of 2μl into the left medial forebrain bundle (6-OHDA - 2.5 µg µl-1 in 0.9% NaCl supplemented with 0.02% ascorbic acid, 1 μL min-1). After the microinjection, the cannula was left in place for two additional minutes to prevent the injected solution’s reflux. At the end of the surgical procedure, the animals were kept warm by a 60W light bulb until full recovery from anaesthesia. The dopamine lesion was confirmed by analysis of tyrosine hydroxylase immunoreactivity (TH-ir) as described before (Padovan-Neto et al., 2015) in the striatum and SNc (Fig. 1).