Oral administration of the KATP channel opener diazoxide ameliorates disease progression in a murine model of multiple sclerosis.

BACKGROUND: Multiple Sclerosis (MS) is an acquired inflammatory demyelinating disorder of the central nervous system (CNS) and is the leading cause of nontraumatic disability among young adults. Activated microglial cells are important effectors of demyelination and neurodegeneration, by secreting cytokines and others neurotoxic agents. Previous studies have demonstrated that microglia expresses ATP-sensitive potassium (KATP) channels and its pharmacological activation can provide neuroprotective and anti-inflammatory effects. In this study, we have examined the effect of oral administration of KATP channel opener diazoxide on induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. METHODS: Anti-inflammatory effects of diazoxide were studied on lipopolysaccharide (LPS) and interferon gamma (IFNγ)-activated microglial cells. EAE was induced in C57BL/6J mice by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35₋55). Mice were orally treated daily with diazoxide or vehicle for 15 days from the day of EAE symptom onset. Treatment starting at the same time as immunization was also assayed. Clinical signs of EAE were monitored and histological studies were performed to analyze tissue damage, demyelination, glial reactivity, axonal loss, neuronal preservation and lymphocyte infiltration. RESULTS: Diazoxide inhibited in vitro nitric oxide (NO), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) production and inducible nitric oxide synthase (iNOS) expression by activated microglia without affecting cyclooxygenase-2 (COX-2) expression and phagocytosis. Oral treatment of mice with diazoxide ameliorated EAE clinical signs but did not prevent disease. Histological analysis demonstrated that diazoxide elicited a significant reduction in myelin and axonal loss accompanied by a decrease in glial activation and neuronal damage. Diazoxide did not affect the number of infiltrating lymphocytes positive for CD3 and CD20 in the spinal cord. CONCLUSION: Taken together, these results demonstrate novel actions of diazoxide as an anti-inflammatory agent, which might contribute to its beneficial effects on EAE through neuroprotection. Treatment with this widely used and well-tolerated drug may be a useful therapeutic intervention in ameliorating MS disease.

Effects of Low Phytanic Acid-Concentrated DHA on Activated Microglial Cells: Comparison with a Standard Phytanic Acid-Concentrated DHA.

Docosahexaenoic acid (DHA, 22:6 n-3) is an essential omega-3 (ω-3) long chain polyunsaturated fatty acid of neuronal membranes involved in normal growth, development, and function. DHA has been proposed to reduce deleterious effects in neurodegenerative processes. Even though, some inconsistencies in findings from clinical and pre-clinical studies with DHA could be attributed to the presence of phytanic acid (PhA) in standard DHA treatments. Thus, the aim of our study was to analyze and compare the effects of a low PhA-concentrated DHA with a standard PhA-concentrated DHA under different neurotoxic conditions in BV-2 activated microglial cells. To this end, mouse microglial BV-2 cells were stimulated with either lipopolysaccharide (LPS) or hydrogen peroxide (H2O2) and co-incubated with DHA 50 ppm of PhA (DHA (PhA:50)) or DHA 500 ppm of PhA (DHA (PhA:500)). Cell viability, superoxide anion (O2-) production, Interleukin 6 (L-6), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), glutathione peroxidase (GtPx), glutathione reductase (GtRd), Caspase-3, and the brain-derived neurotrophic factor (BDNF) protein expression were explored. Low PhA-concentrated DHA protected against LPS or H2O2-induced cell viability reduction in BV-2 activated cells and O2- production reduction compared to DHA (PhA:500). Low PhA-concentrated DHA also decreased COX-2, IL-6, iNOS, GtPx, GtRd, and SOD-1 protein expression when compared to DHA (PhA:500). Furthermore, low PhA-concentrated DHA increased BDNF protein expression in comparison to DHA (PhA:500). The study provides data supporting the beneficial effect of low PhA-concentrated DHA in neurotoxic injury when compared to a standard PhA-concentrated DHA in activated microglia.

Natural Docosahexaenoic Acid in the Triglyceride Form Attenuates In Vitro Microglial Activation and Ameliorates Autoimmune Encephalomyelitis in Mice.

Many neurodegenerative diseases are associated, at least in part, to an inflammatory process in which microglia plays a major role. The effect of the triglyceride form of the omega-3 polyunsaturated fatty acid docosahexaenoic acid (TG-DHA) was assayed in vitro and in vivo to assess the protective and anti-inflammatory activity of this compound. In the in vitro study, BV-2 microglia cells were previously treated with TG-DHA and then activated with Lipopolysaccharide (LPS) and Interferon-gamma (IFN-γ). TG-DHA treatment protected BV-2 microglia cells from oxidative stress toxicity attenuating NO production and suppressing the induction of inflammatory cytokines. When compared with DHA in the ethyl-ester form, a significant difference in the ability to inhibit NO production in favor of TG-DHA was observed. TG-DHA inhibited significantly splenocyte proliferation but isolated CD4+ lymphocyte proliferation was unaffected. In a mice model of autoimmune encephalomyelitis (EAE), 250 mg/kg/day oral TG-DHA treatment was associated with a significant amelioration of the course and severity of the disease as compared to untreated animals. TG-DHA-treated EAE mice showed a better weight profile, which is a symptom related to a better course of encephalomyelitis. TG-DHA may be a promising therapeutic agent in neuroinflammatory processes and merit to be more extensively studied in human neurodegenerative disorders.

Neuronal activity regulates correlated network properties of spontaneous calcium transients in astrocytes in situ.

Spontaneous neuronal activity is essential to neural development. Until recently, neurons were believed to be the only excitable cells to display spontaneous activity. However, cultured astrocytes and, more recently, astrocytes in situ are now known to exhibit spontaneous Ca2+ transients. Here we used Ca2+ imaging of astrocytes from transgenic mice for the simultaneous monitoring of [Ca2+]i changes in large numbers of astrocytes. We found that spontaneous activity is a common property of most brain astrocytes that is lost in response to a lesion. These spontaneous [Ca2+]i oscillations require extracellular and intracellular Ca2+. Moreover, network analysis revealed that most astrocytes formed correlated networks of dozens of these cells, which were synchronous with both astrocytes and neurons. We found that decreasing spontaneous [Ca2+]i transients in neurons by TTX does not alter the number of active astrocytes, although it impairs their synchronous network activity. Conversely, bicuculline-induced epileptic patterns of [Ca2+]i transients in neurons cause an increase in the number of active astrocytes and in their network synchrony. Furthermore, activation of non-NMDA and NMDA ionotropic glutamate receptors is required to correlate astrocytic networks. These results show that spontaneous activity in astrocytes and neurons is patterned into correlated neuronal/astrocytic networks in which neuronal activity regulates the network properties of astrocytes. This network activity may be essential for neural development and synaptic plasticity.

K(ATP) channel opener diazoxide prevents neurodegeneration: a new mechanism of action via antioxidative pathway activation.

Pharmacological modulation of ATP-sensitive potassium channels has become a promising new therapeutic approach for the treatment of neurodegenerative diseases due to their role in mitochondrial and cellular protection. For instance, diazoxide, a well-known ATP-sensitive potassium channel activator with high affinity for mitochondrial component of the channel has been proved to be effective in animal models for different diseases such as Alzheimer's disease, stroke or multiple sclerosis. Here, we analyzed the ability of diazoxide for protecting neurons front different neurotoxic insults in vitro and ex vivo. Results showed that diazoxide effectively protects NSC-34 motoneurons from glutamatergic, oxidative and inflammatory damage. Moreover, diazoxide decreased neuronal death in organotypic hippocampal slice cultures after exicitotoxicity and preserved myelin sheath in organotypic cerebellar cultures exposed to pro-inflammatory demyelinating damage. In addition, we demonstrated that one of the mechanisms of actions implied in the neuroprotective role of diazoxide is mediated by the activation of Nrf2 expression and nuclear translocation. Nrf2 expression was increased in NSC-34 neurons in vitro as well as in the spinal cord of experimental autoimmune encephalomyelitis animals orally administered with diazoxide. Thus, diazoxide is a neuroprotective agent against oxidative stress-induced damage and cellular dysfunction that can be beneficial for diseases such as multiple sclerosis.

Correction: KATP Channel Opener Diazoxide Prevents Neurodegeneration: A New Mechanism of Action via Antioxidative Pathway Activation.

[This corrects the article DOI: 10.1371/journal.pone.0075189.].

Age-dependent spontaneous hyperexcitability and impairment of GABAergic function in the hippocampus of mice lacking trkB.

Patterned intrinsic network activity plays a central role in shaping immature neuronal networks into functional circuits. However, the long-lasting signals that regulate spontaneous activity of developing circuits have not been identified. Here we study the net impact of TrkB signaling on early network activity of identified neuronal populations by analyzing postnatal hippocampi from trkB null mice. Ca2+ imaging showed that pyramidal neurons of trkB-/- mice displayed a decrease in intrinsic synchronous activity in neonatal animals but an increase in juveniles. Strikingly, alterations in network activity in trkB-/- hippocampus were associated with an aberrant induction of the transcription factor Fos. In contrast to pyramidal neurons, spontaneous [Ca2+]i oscillations in trkB-/- interneurons were consistently impaired throughout postnatal development. Moreover, the number of GABAergic synapses and the expression levels of GAD65 and KCC2 were decreased in mutant hippocampi, indicating that pre- and post-synaptic GABAergic components were impaired in trkB-/- mice. Finally, the partial blockade of GABA(A) receptor in postnatal slices revealed that mutant hippocampi displayed an increased susceptibility to network hyperexcitability. These results indicate that the lack of TrkB signaling during development impairs GABAergic neurotransmission, thereby leading to an age-dependent decrease followed by an increase in the intrinsic excitability of neuronal circuits. Furthermore, the present study indicates that long-lasting TrkB signaling may contribute to the construction of CNS circuits by modulating patterns of spontaneous [Ca2+]i oscillations.

Spontaneous network activity of cerebellar granule neurons: impairment by in vivo chronic cannabinoid administration.

Synchronized activity of neuronal networks has been proposed to be essential for cerebellar function. To examine the occurrence and organization of spontaneous neuronal activity in the cerebellum in vivo, we imaged mouse cerebellar slices loaded with the intracellular Ca2+ concentration indicator, fura-2. Recordings were then analysed statistically to identify correlated network activity. Ca2+ imaging revealed consistent spontaneous correlated network activity of granule cells (GC), which often occurred in clusters of coactivated GC. The number of spontaneously active GC, their activation frequency and correlation, were controlled by glutamate and GABA ionotropic receptors. These findings indicate that distinctive patterns of correlated activity between GC networks may be relevant for cerebellar circuit function. Cannabinoid antagonist-precipitated delta9-tetrahydrocannabinol (THC) withdrawal impaired motor coordination. Given that the cerebellum has been suggested recently to be a main substrate for cannabinoid withdrawal, we used imaging of spontaneous network activity to examine whether GC, which contain CB1 cannabinoid receptors, respond to chronic THC treatment and withdrawal. Acute administration of THC had no effect on patterns of spontaneous GC network activity. In contrast, chronic THC administration severely impaired GC activity and network coordination. Incubation of cerebellar slices, from chronically THC-treated mice, with the cannabinoid antagonist, SR141716A increased the number and network correlation of active GC. These data provide physiological evidence of the involvement of cerebellar circuits in the adaptive changes occurring during chronic THC exposure and withdrawal.