Sustained anti-inflammatory effects of TGF-ß1 on microglia/macrophages.

Ischemic brain injuries caused release of damage-associated molecular patterns (DAMPs) that activate microglia/macrophages (MG/MPs) by binding to Toll-like receptors. Using middle cerebral artery transiently occluded rats, we confirmed that MG/MPs expressed inducible nitric oxide synthase (iNOS) on 3days after reperfusion (dpr) in ischemic rat brain. iNOS expression almost disappeared on 7dpr when transforming growth factor-ß1 (TGF-ß1) expression was robustly increased. After transient incubation with TGF-ß1 for 24h, rat primary microglial cells were incubated with lipopolysaccharide (LPS) and released NO level was measured. The NO release was persistently suppressed even 72h after removal of TGF-ß1. The sustained TGF-ß1 effects were not attributable to microglia-derived endogenous TGF-ß1, as revealed by TGF-ß1 knockdown and in vitro quantification studies. Then, boiled supernatants prepared from ischemic brain tissues showed the similar sustained inhibitory effects on LPS-treated microglial cells that were prevented by the TGF-ß1 receptor-selective blocker SB525334. After incubation with TGF-ß1 for 24h and its subsequent removal, LPS-induced phosphorylation of IκB kinases (IKKs), IκB degradation, and NFκB nuclear translocation were inhibited in a sustained manner. SB525334 abolished all these effects of TGF-ß1. In consistent with the in vitro results, phosphorylated IKK-immunoreactivity was abundant in MG/MPs in ischemic brain lesion on 3dpr, whereas it was almost disappeared on 7dpr. The findings suggest that abundantly produced TGF-ß1 in ischemic brain displays sustained anti-inflammatory effects on microglial cells by persistently inhibiting endogenous Toll-like receptor ligand-induced IκB degradation.

Modafinil alleviates levodopa-induced excessive nighttime sleepiness and restores monoaminergic systems in a nocturnal animal model of Parkinson's disease.

Treatment with dopaminergic agents result excessive daytime sleepiness (EDS) and some studies have shown the benefit of using modafinil for treating excessive daytime sleepiness of Parkinson's disease (PD) patient. We investigated whether modafinil have ameliorative properties against levodopa induced excessive nighttime sleepiness (ENS) in MPTP-treated murine nocturnal PD model. Our EEG analyses of whole day recordings revealed that modafinil reduce ENS of this nocturnal PD models with levodopa medications. Therefore, we investigated whether, modafinil post-treatment followed by MPTP shows any effect on monoamine contents of brain and found to robustly increased noradrenaline (NA) concentration of MPTP treated mice. Modafinil post-treatment, in neurorestorative context (5 days post-lesion) led to increased striatal dopamine (DA) concentrations of MPTP-treated mice. Here, we first confirmed that modafinil ameliorates levodopa induced excessive sleepiness and restores monoaminergic systems. The arousal and anti-parkinsonian effects displayed by modafinil indicate that in combination with dopaminergic agents, modafinil co-administration may be worthwhile in trying to suppress the excessive daytime sleepiness and progressive dopaminergic neuron loss in PD.

Microglia may compensate for dopaminergic neuron loss in experimental Parkinsonism through selective elimination of glutamatergic synapses from the subthalamic nucleus.

Parkinson's disease (PD) symptoms do not become apparent until most dopaminergic neurons in the substantia nigra pars compacta (SNc) degenerate, suggesting that compensatory mechanisms play a role. Here, we investigated the compensatory involvement of activated microglia in the SN pars reticulata (SNr) and the globus pallidus (GP) in a 6-hydroxydopamine-induced rat hemiparkinsonism model. Activated microglia accumulated more markedly in the SNr than in the SNc in the model. The cells had enlarged somata and expressed phagocytic markers CD68 and NG2 proteoglycan in a limited region of the SNr, where synapsin I- and postsynaptic density 95-immunoreactivities were reduced. The activated microglia engulfed pre- and post-synaptic elements, including NMDA receptors into their phagosomes. Cells in the SNr and GP engulfed red fluorescent DiI that was injected into the subthalamic nucleus (STN) as an anterograde tracer. Rat primary microglia increased their phagocytic activities in response to glutamate, with increased expression of mRNA encoding phagocytosis-related factors. The synthetic glucocorticoid dexamethasone overcame the stimulating effect of glutamate. Subcutaneous single administration of dexamethasone to the PD model rats suppressed microglial activation in the SNr, resulting in aggravated motor dysfunctions, while expression of mRNA encoding glutamatergic, but not GABAergic, synaptic elements increased. These findings suggest that microglia in the SNr and GP become activated and selectively eliminate glutamatergic synapses from the STN in response to increased glutamatergic activity. Thus, microglia may be involved in a negative feedback loop in the indirect pathway of the basal ganglia to compensate for the loss of dopaminergic neurons in PD brains.

Effects of hypnotic bromovalerylurea on microglial BV2 cells.

An old sedative and hypnotic bromovalerylurea (BU) has anti-inflammatory effects. BU suppressed nitric oxide (NO) release and proinflammatory cytokine expression by lipopolysaccharide (LPS)-treated BV2 cells, a murine microglial cell line. However, BU did not inhibit LPS-induced nuclear translocation of nuclear factor-κB and subsequent transcription. BU suppressed LPS-induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) and expression of interferon regulatory factor 1 (IRF1). The Janus kinase 1 (JAK1) inhibitor filgotinib suppressed the NO release much more weakly than that of BU, although filgotinib almost completely prevented LPS-induced STAT1 phosphorylation. Knockdown of JAK1, STAT1, or IRF1 did not affect the suppressive effects of BU on LPS-induced NO release by BV2 cells. A combination of BU and filgotinib synergistically suppressed the NO release. The mitochondrial complex I inhibitor rotenone, which did not prevent STAT1 phosphorylation or IRF1 expression, suppressed proinflammatory mediator expression less significantly than BU. BU and rotenone reduced intracellular ATP (iATP) levels to a similar extent. A combination of rotenone and filgotinib suppressed NO release by LPS-treated BV2 cells as strongly as BU. These results suggest that anti-inflammatory actions of BU may be attributable to the synergism of inhibition of JAK1/STAT1-dependent pathways and reduction in iATP level.

Pramipexole upregulates dopamine receptor D2 and D3 expression in rat striatum.

Randomized clinical trials have shown that pramipexole has an antidepressant effect in patients with Parkinson's disease. We investigated the comparative efficacy of pramipexole toward dopamine receptor D(2) and D(3) expression in rat brain. Groups of rats were treated subacutely with pramipexole (1 mg/kg), imipramine (10 mg/kg), or bromocriptine (5 mg/kg), with appropriate controls. Using real-time RT-PCR and immunoblotting, dopamine receptor D(2) and D(3) expression was up-regulated in the striatum following pramipexole treatment, while imipramine and bromocriptine had no significant effects. These findings support that pramipexole exerts additional therapeutic benefits such as decreasing depression by increasing dopamine receptor D(3) expression in the striatum.

Utility of Rectus Abdominis Ultrasound as a Respiratory Function Test for Amyotrophic Lateral Sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) causes deterioration of respiratory function. Muscle weakness of the orbicularis oris interferes with the accurate assessment of respiratory function using spirometry. Reduced forced vital capacity (FVC) is an indicator that helps determine the appropriate timing to provide noninvasive ventilation (NIV) for the survival of ALS patients. We employed ultrasonography to evaluate changes in respiratory function by measuring the thickness of the rectus abdominis (RA) muscle as a possible alternative to spirometry. METHODS: Sixteen subjects with ALS were included in this study. The thickness of RA muscles was measured using ultrasonography, and respiratory fluctuations, such as vital capacity (VC), FVC, FEV1, percentage of predicted VC (%VC), percentage of predicted FVC (%FVC), percentage of predicted FEV1 (%FEV1), and FEV1/FVC, were evaluated using spirometry. RESULTS: Sixteen subjects underwent assessment by ultrasonography. A positive correlation was observed between the percent change in RA muscle thickness evaluated from maximal expiration to maximal inspiration and %VC (P = .001), %FVC (P = .001), FEV1 (P = .009), and %FEV1 (P = .02). CONCLUSIONS: RA ultrasonography was useful for predicting a reduction in VC in subjects with ALS and may help determine the best timing for introducing NIV.

Impact of Intestinal Bacteria on Levodopa Pharmacokinetics in LCIG Therapy.

Background: Levodopa-carbidopa intestinal gel (LCIG) therapy is used in advanced Parkinson's disease (PD) and consists of continuous administration of levodopa directly into the jejunum through a percutaneous endoscopic gastro-jejunal (PEG-J) tube. Recently, the metabolism of levodopa by Enterococcus faecalis (E. faecalis) has been reported. Intestinal bacteria can also affect this therapy. Objectives: To investigate intestinal bacteria and examine its impact on levodopa blood concentration in patients with PD receiving LCIG therapy. Methods: We enrolled 6 patients receiving LCIG therapy in our department. After PEG-J tube replacement, intestinal bacteria were collected from the tip of the tube and were identified using culture and polymerase chain reaction (PCR) tests. Moreover, the presence of tyrosine decarboxylase, which metabolizes levodopa, was also confirmed by PCR test. The ability of these bacteria to metabolize levodopa was confirmed in vitro. Levodopa blood concentrations were also examined before PEG-J tube replacement. Results: Bacteria were detected in all 6 patients. E. faecalis was present in 4 patients. Moreover, tyrosine decarboxylase was detected in 2 patients. The identified bacteria displayed in vitro metabolization to dopamine in the 4 E. faecalis positive samples. The addition of carbidopa did not inhibit the metabolism of levodopa. However, there was no difference in the mean blood concentration of levodopa, regardless of the presence of E. faecalis. Conclusions: We found bacteria, including E. faecalis in the PEG-J tube. We observed levodopa metabolism in vitro, but there was no association with levodopa blood concentration. The effect of intestinal bacteria may be limited in patients receiving LCIG therapy.

Zonisamide attenuates MPTP neurotoxicity in marmosets.

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induces parkinsonism in humans and animals. The effects of zonisamide on dopamine neurons were studied in MPTP-treated common marmosets (Callithrix jacchus). Groups of animals (n = 3) were treated with MPTP (2.5 mg/kg, every 24 h x 3); MPTP plus zonisamide (40 mg/kg administered 1 h before each MPTP dose); MPTP plus selegiline (a known MAO-B inhibitor) (2 mg/kg administered 1 h before each MPTP dose); and saline controls. An immunohistochemical study of the substantia nigra was performed 14 days after MPTP treatment in each group. MPTP reduced the mean number of tyrosine hydroxylase (TH)-positive neurons to 10% of the normal control group and mean cell size was significantly (P < 0.001) reduced from 424 to 159 µm². In the group pre-treated with zonisamide, the mean number of TH-positive neurons was reduced to 26% of that in the normal control group and the mean neuron size was significantly (P < 0.05) increased from 159 to 273 µm² compared with the group treated with MPTP alone. Moreover, in the group pre-treated with selegiline, the mean number of TH-positive neurons was 47% of that in the normal control group and the mean neuron size was increased significantly (P < 0.01) from 159 to 319 µm² compared to the group treated with MPTP alone. This observation suggests that zonisamide reduces MPTP toxicity.

The hypnotic bromovalerylurea ameliorates 6-hydroxydopamine-induced dopaminergic neuron loss while suppressing expression of interferon regulatory factors by microglia.

The low molecular weight organic compound bromovalerylurea (BU) has long been used as a hypnotic/sedative. In the present study, we found that BU suppressed mRNA expression of proinflammatory factors and nitric oxide release in lipopolysaccharide (LPS)-treated rat primary microglial cell cultures. BU prevented neuronal degeneration in LPS-treated neuron-microglia cocultures. The anti-inflammatory effects of BU were as strong as those of a synthetic glucocorticoid, dexamethasone. A rat hemi-Parkinsonian model was prepared by injecting 6-hydroxydopamine into the right striatum. BU was orally administered to these rats for 7 days, which ameliorated the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and alleviated motor deficits. BU suppressed the expression of mRNAs for interferon regulatory factors (IRFs) 1, 7 and 8 in the right (lesioned) ventral midbrain as well as those for proinflammatory mediators. BU increased mRNA expression of various neuroprotective factors, including platelet-derived growth factor and hepatocyte growth factor, but it did not increase expression of alternative activation (M2) markers. In microglial culture, BU suppressed the LPS-induced increase in expression of IRFs 1 and 8, and it reduced LPS-induced phosphorylation of JAK1 and STATs 1 and 3. Knockdown of IRFs 1 and 8 suppressed LPS-induced NO release by microglial cells. These results suggest that suppression of microglial IRF expression by BU prevents neuronal cell death in the injured brain region, where microglial activation occurs. Because many Parkinsonian patients suffer from sleep disorders, BU administration before sleep may effectively ameliorate neurological symptoms and alleviate sleep dysfunction.

Zonisamide-induced long-lasting recovery of dopaminergic neurons from MPTP-toxicity.

Zonisamide is an antiepileptic drug that also improves the cardinal symptoms of Parkinson's disease. This study investigated the effects of zonisamide on dopaminergic neuronal degeneration in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Six groups of mice were treated as follows: 1) normal saline; 2) MPTP, 15 mg/kg×4 every 2h; 3) MPTP and zonisamide, 40 mg/kg×1, 1h after the last MPTP dose; 4) MPTP and zonisamide, 1 day after the last dose of MPTP; 5) MPTP and zonisamide, 1h before the first MPTP dose; and 6) zonisamide, 40 mg/kg. MPTP-treatment decreased the contents of dopamine as well as the number and area of tyrosine hydroxylase (TH)-positive neurons. Concurrent treatment of mice with zonisamide and MPTP did not show any inhibition of the toxic effect of MPTP towards dopamine contents at 1 week after treatment but it increased the number and area of TH-positive neurons compared to the MPTP-treated group. Surviving TH-positive neurons had recovery of dopamine production after several weeks. Moreover, zonisamide increased the number of S100ß-positive and glial fibrillary acidic protein (GFAP)-positive astrocytes and dopamine turnover. These results suggest that zonisamide acts as a neuro-protectant against MPTP-induced dopaminergic neuronal degeneration as shown by an increase of TH-positive neurons and this may be mediated by increased S100ß secretion.

A cytokine mixture of GM-CSF and IL-3 that induces a neuroprotective phenotype of microglia leading to amelioration of (6-OHDA)-induced Parkinsonism of rats.

Dopamine (DA) agonists are widely used as primary treatments for Parkinson's disease. However, they do not prevent progressive degeneration of dopaminergic neurons, the central pathology of the disease. In this study, we found that subcutaneous injection of a cytokine mixture containing granulocyte macrophage colony-stimulating factor and interleukin-3 (IL-3) markedly suppressed dopaminergic neurodegeneration in 6-hydroxydopamine-lesioned rats, an animal model of Parkinson's disease. The cytokine mixture suppressed the decrease of DA content in the striatum, and ameliorated motor function in the lesioned rats. In response to the cytokine injection, dopaminergic neurons in the substantia nigra pars compacta increased expression of the antiapoptotic protein Bcl-xL. Microglial activation in the pars compacta was evident in both the saline- and cytokine-injected rats. However, the cytokine mixture suppressed expression of the proinflammatory cytokines IL-1ß and tumor necrosis factors α, and upregulated the neuroprotective factors insulin-like growth factor-1 and hepatocyte growth factor. Similar responses were observed in cultured microglia. Detailed morphometric analyses revealed that NG2 proteoglycan-expressing glial cells increased in the cytokine-injected rats, while astrocytic activation with increased expression of antioxidative factors was evident only in the saline-injected rats. Thus, the present findings show that the cytokine mixture was markedly effective in suppressing neurodegeneration. Its neuroprotective effects may be mediated by increased expression of Bcl-xL in dopaminergic neurons, and the activation of beneficial actions of microglia that promote neuronal survival. Furthermore, this cytokine mixture may have indirect actions on NG2 proteoglycan-expressing glia, whose role may be implicated in neuronal survival.

Zonisamide increases dopamine turnover in the striatum of mice and common marmosets treated with MPTP.

The effect of zonisamide, an antiepileptic agent with anti-parkinsonian effects, was studied on dopamine neurons of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated C57 mice and common marmosets. Groups of mice (n = 8 - 9) were treated with: MPTP (15 mg/kg every 2 h x4); MPTP plus zonisamide (40 mg/kg administered 1 h before each MPTP dose); MPTP plus selegiline (2 mg/kg administered 1 h before the first MPTP dose); zonisamide (40 mg/kg x4); and saline controls. Groups of common marmosets (n = 4 - 6) were treated with: MPTP (2.5 mg/kg every 24 h x3); MPTP plus zonisamide (40 mg/kg administered 1 h before each MPTP dose); MPTP plus selegiline (2 mg/kg administered 1 h before the first MPTP dose); and saline controls. Brain dopamine and its metabolites were determined by HPLC. Dopamine content decreased in the striatum of MPTP-treated mice and monkeys. Co-administration of selegiline inhibited the effect of MPTP and dopamine contents were similar to those of the controls. Co-administration of zonisamide did not inhibit the effect of MPTP on dopamine content, but increased striatal dopamine turnover of animals treated with MPTP plus zonisamide more than in those treated with MPTP alone. MPTP treatment caused a compensatory increase of dopamine turnover in the striatum by remaining neurons. Zonisamide may help dopaminergic neurons by increasing striatal dopamine turnover following MPTP treatment.