Pomalidomide Improves Motor Behavioral Deficits and Protects Cerebral Cortex and Striatum Against Neurodegeneration Through a Reduction of Oxidative/Nitrosative Damages and Neuroinflammation After Traumatic Brain Injury.

Neuronal damage resulting from traumatic brain injury (TBI) causes disruption of neuronal projections and neurotransmission that contribute to behavioral deficits. Cellular generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is an early event following TBI. ROS often damage DNA, lipids, proteins, and carbohydrates while RNS attack proteins. The products of lipid peroxidation 4-hydroxynonenal (4-HNE) and protein nitration 3-nitrotyrosine (3-NT) are often used as indicators of oxidative and nitrosative damages, respectively. Increasing evidence has shown that striatum is vulnerable to damage from TBI with a disturbed dopamine neurotransmission. TBI results in neurodegeneration, oxidative stress, neuroinflammation, neuronal apoptosis, and autophagy in the striatum and contribute to motor or behavioral deficits. Pomalidomide (Pom) is a Food and Drug Administration (FDA)-approved immunomodulatory drug clinically used in treating multiple myeloma. We previously showed that Pom reduces neuroinflammation and neuronal death induced by TBI in rat cerebral cortex. Here, we further compared the effects of Pom in cortex and striatum focusing on neurodegeneration, oxidative and nitrosative damages, as well as neuroinflammation following TBI. Sprague-Dawley rats subjected to a controlled cortical impact were used as the animal model of TBI. Systemic administration of Pom (0.5 mg/kg, intravenous [i.v.]) at 5 h post-injury alleviated motor behavioral deficits, contusion volume at 24 h after TBI. Pom alleviated TBI-induced neurodegeneration stained by Fluoro-Jade C in both cortex and striatum. Notably, Pom treatment reduces oxidative and nitrosative damages in cortex and striatum and is more efficacious in striatum (93% reduction in 4-HNE-positive and 84% reduction in 3-NT-positive neurons) than in cerebral cortex (42% reduction in 4-HNE-positive and 55% reduction in 3-NT-positive neurons). In addition, Pom attenuated microgliosis, astrogliosis, and elevations of proinflammatory cytokines in cortical and striatal tissue. We conclude that Pom may contribute to improved motor behavioral outcomes after TBI through targeting oxidative/nitrosative damages and neuroinflammation.

Neuroprotective effects of pifithrin-α against traumatic brain injury in the striatum through suppression of neuroinflammation, oxidative stress, autophagy, and apoptosis.

Cortical and hippocampal neuronal damages caused by traumatic brain injury (TBI) are associated with motor and cognitive impairments; however, only little attention paid to the striatal damage. It is known that the p53 tumor-suppressor transcription factor participated in TBI-induced secondary brain damage. We investigated how the p53 inactivator pifithrin (PFT)-α affected TBI-induced striatal neuronal damage at 24 h post-injury. Sprague-Dawley rats subjected to a controlled cortical impact were used as TBI models. We observed that p53 mRNA significantly increased, whereas p53 protein expression was distributed predominantly in neurons but not in glia cells in striatum after TBI. PFT-α improved motor deficit following TBI. PFT-α suppressed TBI-induced striatal glial activation and expression of proinflammatory cytokines. PFT-α alleviated TBI-induced oxidative damage TBI induced autophagy was evidenced by increased protein expression of Beclin-1 and shift of microtubule-associated light chain (LC)3-I to LC3-II, and decreased p62. These effects were reduced by PFT-α. Post-injury PFT-α treatment reduced the number of degenerating (FJC-positive) and apoptotic neurons. Our results suggest that PFT-α may provide neuroprotective effects via p53-dependent or -independent mechanisms depending on the cell type and timing after the TBI and can possibly be developed into a novel therapy to ameliorate TBI-induced neuronal damage.

L-Ascorbate Protects Against Methamphetamine-Induced Neurotoxicity of Cortical Cells via Inhibiting Oxidative Stress, Autophagy, and Apoptosis.

Methamphetamine (METH)-induced cell death contributes to the pathogenesis of neurotoxicity; however, the relative roles of oxidative stress, apoptosis, and autophagy remain unclear. L-Ascorbate, also called vitamin (Vit.) C, confers partial protection against METH neurotoxicity via induction of heme oxygenase-1. We further investigated the role of Vit. C in METH-induced oxidative stress, apoptosis, and autophagy in cortical cells. Exposure to lower concentrations (0.1, 0.5, 1 mM) of METH had insignificant effects on ROS production, whereas cells exposed to 5 mM METH exhibited ROS production in a time-dependent manner. We confirmed METH-induced apoptosis (by nuclear morphology revealed by Hoechst 33258 staining and Western blot showing the protein levels of pro-caspase 3 and cleaved caspase 3) and autophagy (by Western blot showing the protein levels of Belin-1 and conversion of microtubule-associated light chain (LC)3-I to LC3-II and autophagosome staining by monodansylcadaverine). The apoptosis as revealed by cleaved caspase-3 expression marked an increase at 18 h after METH exposure while both autophagic markers, Beclin 1 and LC3-II, marked an increase in cells exposed to METH for 6 and 24 h, respectively. Treating cells with Vit. C 30 min before METH exposure time-dependently attenuated the production of ROS. Vitamin C also attenuated METH-induced Beclin 1 and LC3-II expression and METH toxicity. Treatment of cells with Vit. C before METH exposure attenuated the expression of cleaved caspase-3 and reduced the number of METH-induced apoptotic cells. We suggest that the protective effect of Vit. C against METH toxicity might be through attenuation of ROS production, autophagy, and apoptosis.

Therapeutic Potential of Drugs Targeting Pathophysiology of Intracerebral Hemorrhage: From Animal Models to Clinical Applications.

BACKGROUND: Intracerebral hemorrhage (ICH) is one of the most common forms of cerebral hemorrhage, the morbidity and death of ICH is high worldwide. ICH can be spontaneous or caused by hypertension, coagulopathy, angiopathy, head trauma, bleeding disorders, tumors, or drug usage. ICH is the most serious and least treatable form of hemorrhagic stroke, with rapidly increasing hematoma size and often resulting in significant brain injury and long term neurological deficits. Surgical hematoma evacuation remains controversial. The currently therapy is mainly supportive with limited benefit. New therapeutic approaches are desperately needed. METHODS: In this review, we provide an overview of the published literature concerning the pathophysiology leading to the ongoing neurologic damage, Emerging information of the physio-pathologic mechanisms of injury that occur after ICH is available from current animal models. Ideal therapeutic strategies should target on the pathophysiology of ICH. This review summarizes the recent advances in developing pharmaceutical agents in terms of therapeutic targets and effects in pre-clinical and clinical studies. RESULTS: Recent animal and clinical studies have provided important information about the parallel and sequential deleterious mechanisms underlying ICH-induced brain injury and pharmacological agents targeting on these mechanisms. Neuroscientists have paid more attention to novel drug development that target on antioxidants, antiinflammatory, and anti-apoptosis for neuroprotection after ICH. CONCLUSION: Although ICH remains without an approved treatment proven to decrease morbidity and mortality, notable advances in the understanding of ICH pathophysiology and new drug development have been made in the last decade.

Post-traumatic administration of the p53 inactivator pifithrin-α oxygen analogue reduces hippocampal neuronal loss and improves cognitive deficits after experimental traumatic brain injury.

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Neuronal apoptosis in the hippocampus has been detected after TBI. The hippocampal dysfunction may result in cognitive deficits in learning, memory, and spatial information processing. Our previous studies demonstrated that a p53 inhibitor, pifithrin-α oxygen analogue (PFT-α (O)), significantly reduced cortical cell death, which is substantial following controlled cortical impact (CCI) TBI, and improved neurological functional outcomes via anti-apoptotic mechanisms. In the present study, we examined the effect of PFT-α (O) on CCI TBI-induced hippocampal cellular pathophysiology in light of this brain region's role in memory. To investigate whether p53-dependent apoptosis plays a role in hippocampal neuronal loss and associated cognitive deficits and to define underlying mechanisms, SD rats were subjected to experimental CCI TBI followed by the administration of PFT-α or PFT-α (O) (2mg/kg, i.v.) or vehicle at 5h after TBI. Magnetic resonance imaging (MRI) scans were acquired at 24h and 7days post-injury to assess evolving structural hippocampal damage. Fluoro-Jade C was used to stain hippocampal sub-regions, including CA1 and dentate gyrus (DG), for cellular degeneration. Neurological functions, including motor and recognition memory, were assessed by behavioral tests at 7days post injury. p53, p53 upregulated modulator of apoptosis (PUMA), 4-hydroxynonenal (4-HNE), cyclooxygenase-IV (COX IV), annexin V and NeuN were visualized by double immunofluorescence staining with cell-specific markers. Levels of mRNA encoding for caspase-3, p53, PUMA, Bcl-2, Bcl-2-associated X protein (BAX) and superoxide dismutase (SOD) were measured by RT-qPCR. Our results showed that post-injury administration of PFT-α and, particularly, PFT-α (O) at 5h dramatically reduced injury volumes in the ipsilateral hippocampus, improved motor outcomes, and ameliorated cognitive deficits at 7days after TBI, as evaluated by novel object recognition and open-field test. PFT-α and especially PFT-α (O) significantly reduced the number of FJC-positive cells in hippocampus CA1 and DG subregions, versus vehicle treatment, and significantly decreased caspase-3 and PUMA mRNA expression. PFT-α (O), but not PFT-α, treatment significantly lowered p53 and elevated SOD2 mRNA expression. Double immunofluorescence staining demonstrated that PFT-α (O) treatment decreased p53, annexin V and 4-HNE positive neurons in the hippocampal CA1 region. Furthermore, PUMA co-localization with the mitochondrial maker COX IV, and the upregulation of PUMA were inhibited by PFT-α (O) after TBI. Our data suggest that PFT-α and especially PFT-α (O) significantly reduce hippocampal neuronal degeneration, and ameliorate neurological and cognitive deficits in vivo via antiapoptotic and antioxidative properties.

Pomalidomide mitigates neuronal loss, neuroinflammation, and behavioral impairments induced by traumatic brain injury in rat.

BACKGROUND: Traumatic brain injury (TBI) is a global health concern that typically causes emotional disturbances and cognitive dysfunction. Secondary pathologies following TBI may be associated with chronic neurodegenerative disorders and an enhanced likelihood of developing dementia-like disease in later life. There are currently no approved drugs for mitigating the acute or chronic effects of TBI. METHODS: The effects of the drug pomalidomide (Pom), an FDA-approved immunomodulatory agent, were evaluated in a rat model of moderate to severe TBI induced by controlled cortical impact. Post-TBI intravenous administration of Pom (0.5 mg/kg at 5 or 7 h and 0.1 mg/kg at 5 h) was evaluated on functional and histological measures that included motor function, fine more coordination, somatosensory function, lesion volume, cortical neurodegeneration, neuronal apoptosis, and the induction of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6). RESULTS: Pom 0.5 mg/kg administration at 5 h, but not at 7 h post-TBI, significantly mitigated the TBI-induced injury volume and functional impairments, neurodegeneration, neuronal apoptosis, and cytokine mRNA and protein induction. To evaluate underlying mechanisms, the actions of Pom on neuronal survival, microglial activation, and the induction of TNF-α were assessed in mixed cortical cultures following a glutamate challenge. Pom dose-dependently ameliorated glutamate-mediated cytotoxic effects on cell viability and reduced microglial cell activation, significantly attenuating the induction of TNF-α. CONCLUSIONS: Post-injury treatment with a single Pom dose within 5 h significantly reduced functional impairments in a well-characterized animal model of TBI. Pom decreased the injury lesion volume, augmented neuronal survival, and provided anti-inflammatory properties. These findings strongly support the further evaluation and optimization of Pom for potential use in clinical TBI.

Melatonin reverses morphine tolerance by inhibiting microglia activation and HSP27 expression.

AIMS: Melatonin has been reported to attenuate opioid tolerance. In this study, we explored the possible mechanism of melatonin in diminishing morphine tolerance. MAIN METHODS: Two intrathecal (i.t.) catheters were implanted in male Wistar rats for drug delivery. One was linked to a mini-osmotic pump for morphine or saline infusion. On the seventh day, 50µg of melatonin or vehicle was injected through the other catheter instantly after discontinuation of morphine or saline infusion; 3h later, 15µg of morphine or saline was injected. The antinociceptive response was then measured using the tail-flick test every 30min for 120min. KEY FINDINGS: The results showed that chronic morphine infusion elicited antinociceptive tolerance and upregulated heat shock protein 27 (HSP27) expression in the dorsal horn of the rat spinal cord. Melatonin pretreatment partially restored morphine's antinociceptive effect in morphine-tolerant rats and reversed morphine-induced HSP27 upregulation. In addition, chronic morphine infusion induced microglial cell activation and was reversed by melatonin treatment. SIGNIFICANCE: The present study provides evidence that melatonin, acting via inhibiting morphine-induced neuroinflammation, can be useful as a therapeutic adjuvant for patients under long-term opioid treatment for pain relief.

Reparixin attenuates neuronal injury in experimental Klebsiella pneumoniae meningoencephalitis through dual effects on neuroprotection and neuroinflammation.

AIMS: Bacterial meningitis causes high mortality and brain damage. The host immune response is associated with brain injury. Chemokine (C-X-C motif) (CXC) chemokines are neutrophil chemoattractants. This study focused on the beneficial effects of intracerebroventricular administration of reparixin, an inhibitor of chemokine (C-X-C motif) receptor (CXCR)1/2, to rats at 2 h following experimental Klebsiella pneumoniae meningoencephalitis. METHODS: We used a previously established meningoencephalitis animal model in which Sprague-Dawley rats were infected by K. pneumoniae. Sham and infected animals were treated with vehicle or reparixin and sacrificed at various time points. Leukocyte infiltration into cerebrospinal fluid (CSF) and brain as well as gene and protein expression of chemokines and receptors, and neuronal apoptosis were examined. Primary cultures of neuron/glia were infected with K. pneumoniae as an in vitro model of meningoencephalitis. RESULTS: Levels of chemokine (C-X-C motif) ligand (CXCL)2 in CSF time-dependently increased markedly as early as 2 h, and peaked at 8 h following infection and were much higher than those in serum collected simultaneously. Reparixin significantly reduced leukocyte infiltration into CSF and brain tissues, clinical illness, and brain cell apoptosis at 24 h. Reparixin reduced the elevated CSF concentrations of chemokines [CXCL1, CXCL2, chemokine (C-C motif) ligand (CCL)2 and CCL5] and proinflammatory cytokines. Reparixin also reduced the expression of mRNA of various chemokines, chemokine receptors and proinflammatory cytokines in infected brain tissues. Using primary cultures that are devoid of leukocytes, we further observed that reparixin attenuated the neuronal, but not microglial cell death after infection. CONCLUSIONS: Reparixin not only reduces amplified inflammation, but also provides direct neuroprotective effects in K. pneumoniae meningoencephalitis.

1,25-Dihydroxyvitamin D3 attenuates endotoxin-induced production of inflammatory mediators by inhibiting MAPK activation in primary cortical neuron-glia cultures.

BACKGROUND: Neuroinflammation occurs in insulted regions of the brain and may be due to reactive oxygen species (ROS), nitric oxide (NO), cytokines, and chemokines produced by activated glia. Excessive production of neurotoxic molecules causes further neuronal damage. Low levels of vitamin D3 are a risk factor for various brain diseases. METHODS: Using the bacterial endotoxin, lipopolysaccharide (LPS), to induce neuroinflammation in primary cortical neuron-glia cultures, we investigated how 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) affected neuroinflammation. RESULTS: LPS (100 ng/ml) induced the accumulation of nitrite and the production of ROS, interleukin (IL)-6, and macrophage inflammatory protein (MIP)-2 in time-dependent manners. Inhibition of p38 and extracellular signal-regulated kinase (ERK) but not c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) by 20 µM of SB203580, PD98059, and SP600125, significantly reduced LPS-induced ROS production, NO accumulation, and inducible NO synthase (iNOS) expression, respectively. LPS-induced IL-6 and MIP-2 were significantly attenuated by inhibition of p38, ERK, and JNK MAPK. Cotreatment with 1,25(OH)2D3 attenuated LPS-induced ROS production, NO accumulation, and iNOS expression in concentration-dependent manners. 1,25(OH)2D3 also reduced LPS-induced production of IL-6 and MIP-2. Similarly, iNOS, IL-6, and MIP-2 mRNA expression in cells treated with LPS significantly increased, whereas this effect was attenuated by 1,25(OH)2D3. Moreover, LPS-induced phosphorylation of p38, ERK, and JNK MAPK was significantly inhibited by 1,25(OH)2D3. CONCLUSIONS: Our findings indicate that 1,25(OH)2D3 reduced the LPS-stimulated production of inflammatory molecules in neuron-glia cultures by inhibiting MAPK pathways and the production of downstream inflammatory molecules. We suggest that 1,25(OH)2D3 can be used to alleviate neuroinflammation in various brain injuries.

L-ascorbate attenuates the endotoxin-induced production of inflammatory mediators by inhibiting MAPK activation and NF-κB translocation in cortical neurons/glia Cocultures.

In response to acute insults to the central nervous system, such as pathogen invasion or neuronal injuries, glial cells become activated and secrete inflammatory mediators such as nitric oxide (NO), cytokines, and chemokines. This neuroinflammation plays a crucial role in the pathophysiology of chronic neurodegenerative diseases. Endogenous ascorbate levels are significantly decreased among patients with septic encephalopathy. Using the bacterial endotoxin lipopolysaccharide (LPS) to induce neuroinflammation in primary neuron/glia cocultures, we investigated how L-ascorbate (vitamin C; Vit. C) affected neuroinflammation. LPS (100 ng/ml) induced the expression of inducible NO synthase (iNOS) and the production of NO, interleukin (IL)-6, and macrophage inflammatory protein-2 (MIP-2/CXCL2) in a time-dependent manner; however, cotreatment with Vit. C (5 or 10 mM) attenuated the LPS-induced iNOS expression and production of NO, IL-6, and MIP-2 production. The morphological features revealed after immunocytochemical staining confirmed that Vit. C suppressed LPS-induced astrocytic and microglial activation. Because Vit. C can be transported into neurons and glia via the sodium-dependent Vit. C transporter-2, we examined how Vit. C affected LPS-activated intracellular signaling in neuron/glia cocultures. The results indicated the increased activation (caused by phosphorylation) of mitogen-activated protein kinases (MAPKs), such as p38 at 30 min and extracellular signal-regulated kinases (ERKs) at 180 min after LPS treatment. The inhibition of p38 and ERK MAPK suppressed the LPS-induced production of inflammatory mediators. Vit. C also inhibited the LPS-induced activation of p38 and ERK. Combined treatments of Vit. C and the inhibitors of p38 and ERK yielded no additional inhibition compared with using the inhibitors alone, suggesting that Vit. C functions through the same signaling pathway (i.e., MAPK) as these inhibitors. Vit. C also reduced LPS-induced IκB-α degradation and NF-κB translocation. Thus, Vit. C suppressed the LPS-stimulated production of inflammatory mediators in neuron/glia cocultures by inhibiting the MAPK and NF-κB signaling pathways.

The effects of electrode spacing on the performance of microbial fuel cells under different substrate concentrations.

In this study, the electricity generation and organic removal in microbial fuel cells (MFCs) were examined for electrode spacing (ES) covering 5.8, 10.2, 15.1, and 19.5 cm, and for each ES the MFCs were discharged with a series of influent substrates (COD(in)). Results indicate that organic removal was related to COD(in) but not to ES. Best chemical oxygen demand (COD) removals of 64-71% could be achieved at COD(in) around 100 mg COD/L (0.11-0.14 kg COD/m(3)-day). Best power output 3.32 mW/m(2) occurred at ES 5.8 cm and nominal COD(in) 300 mg COD/L. For every ES, the relationship of electricity generation to local substrate near anode (COD(ad)) could be adequately modeled by Monod-type kinetics. The estimated kinetic constants involve maximum current production, I(max), 15.3-19.6 mA/m(2); maximum attainable power output, P(p,max), 4.0-2.5 mW/m(2); half-saturation constant of current, K(si), 22-30 mg COD/L; and half-saturation constant of power, K(sp), 24-90 mg COD/L. This study reveals that the control over ES for improving electricity generation is dependent on the level of COD(ad), which profoundly affects the optimal design of electrode placement.

L-ascorbate attenuates methamphetamine neurotoxicity through enhancing the induction of endogenous heme oxygenase-1.

Methamphetamine (METH) is a drug of abuse which causes neurotoxicity and increased risk of developing neurodegenerative diseases. We previously found that METH induces heme oxygenase (HO)-1 expression in neurons and glial cells, and this offers partial protection against METH toxicity. In this study, we investigated the effects of l-ascorbate (vitamin C, Vit. C) on METH toxicity and HO-1 expression in neuronal/glial cocultures. Cell viability and damage were evaluated by 3-(4,5-dimethylthianol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release, respectively. Neuronal and glial localization of HO-1 were identified by double immunofluorescence staining. Reactive oxygen species (ROS) production was measured using the fluorochrome 2',7'-dichlorofluorescin diacetate. HO-1 mRNA and protein expression were examined by RT-qPCR and Western blotting, respectively. Results show that Vit. C induced HO-1 mRNA and protein expressions in time- and concentration-dependent manners. Inhibition of p38 mitogen-activated protein kinase (MAPK) but not extracellular signal-regulated kinase (ERK) significantly blocked induction of HO-1 by Vit. C. HO-1 mRNA and protein expressions were significantly elevated by a combination of Vit. C and METH, compared to either Vit. C or METH alone. Pretreatment with Vit. C enhanced METH-induced HO-1 expression and attenuated METH-induced ROS production and neurotoxicity. Pharmacological inhibition of HO activity abolished suppressive effects of Vit. C on METH-induced ROS production and attenuated neurotoxicity. We conclude that induction of HO-1 expression contributes to the attenuation of METH-induced ROS production and neurotoxicity by Vit. C. We suggest that HO-1 induction by Vit. C may serve as a strategy to alleviate METH neurotoxicity.

Resveratrol regulates N-methyl-D-aspartate receptor expression and suppresses neuroinflammation in morphine-tolerant rats.

BACKGROUND: In the present study, we examined the effects and mechanisms of the Chinese herb resveratrol on attenuation of morphine tolerance in rats. METHODS: Male Wistar rats were implanted with 2 intrathecal catheters; one catheter was connected to a mini-osmotic pump, used for either morphine (15 µg/h) or saline (1 µL/h) infusion for 5 days. On day 5, resveratrol (7.5, 15, 30, or 60 µg), dimethyl sulfoxide (5 µL), or saline (5 µL) was injected via the other catheter immediately after the discontinued morphine infusion. Three hours later, intrathecal morphine (15 µg in 5 µL saline) was given. All rats received the nociceptive tail-flick test every 30 minutes for 120 minutes after the morphine challenge. RESULTS: Long-term morphine infusion induced antinociceptive tolerance and up-regulated N-methyl-D-aspartate receptor (NMDAR) subunit NR1 and NR2B expression in the synaptosome fraction of the tolerant spinal cord dorsal horn. Resveratrol pretreatment provided a significant antinociceptive effect of morphine in morphine-tolerant rats, and it was associated with reversal of the up-regulated NR1 and NR2B subunits in the synaptosome fraction of morphine-tolerant rat spinal cords. NR1/NR2B-specific antagonist ifenprodil treatment produced a similar effect as that of resveratrol. Furthermore, an increase of postsynaptic density-95/NR1/NR2B complex immunoprecipitation in morphine-tolerant rat spinal cord was also inhibited by resveratrol pretreatment. Moreover, chronic morphine infusion activated glial cells with an increase of proinflammatory cytokine tumor necrosis factor-α, interleukin-1ß, and interleukin-6 mRNA expression in morphine-tolerant rat spinal cords and these effects were suppressed by resveratrol pretreatment before the morphine challenge. CONCLUSIONS: Resveratrol attenuates morphine tolerance by inhibiting neuroinflammation and down-regulating NMDAR NR1 and NR2B subunit expression. Resveratrol regulates the NMDAR expression, which might be involved in a loss of scaffolding postsynaptic density-95 protein.

Amitriptyline attenuates astrocyte activation and morphine tolerance in rats: role of the PSD-95/NR1/nNOS/PKCγ signaling pathway.

The tricyclic antidepressant amitriptyline binds with high affinity to N-methyl-d-aspartate receptors (NMDARs) and inhibits NMDAR-mediated events. Activation of the postsynaptic density protein-95 (PSD-95)/NMDAR-mediated downstream signaling cascade, including neuronal nitric oxide synthase (nNOS) and protein kinase gamma (PKCγ), has been shown to be involved in morphine tolerance. The present study examined the potential effect of amitriptyline on chronic morphine infusion-induced spinal PSD-95/NMDAR/nNOS/PKCγ signaling in morphine tolerance. Male Wistar rats were implanted with an intrathecal catheter and received an intrathecal infusion of saline or amitriptyline (15 µg/h), morphine+saline (tolerance induction, 15 µg/h), or morphine+amitriptyline for 5 days. Co-administration of amitriptyline with morphine not only preserved the antinociceptive effect of morphine, but also attenuated astrocyte activation in the rat spinal cord dorsal horn. On day 5 after drug infusion, increased expression and phosphorylation of spinal membrane NMDAR NR1 subunit and expression of PSD-95 were observed following chronic morphine infusion and these effects were attenuated by amitriptyline co-infusion. Upregulation of NMDAR-induced intracellular nNOS expression was also inhibited by amitriptyline co-infusion in chronic morphine-infused rats. Furthermore, amitriptyline co-infusion significantly inhibited morphine-induced PKCγ expression in both the cytosol and membrane of spinal neurons. These findings suggest that the attenuation of morphine tolerance caused by amitriptyline is due to downregulation of NMDAR NR1 subunit expression in the synaptosomal membrane accompanied by decreased expression of the scaffolding protein PSD-95. The effects of amitriptyline in attenuating astrocyte activation and reversing tolerance to morphine may be due, at least in part, to inhibition of the PSD-95/NMDAR NR1/nNOS/PKCγ signaling cascade.

Methamphetamine induces heme oxygenase-1 expression in cortical neurons and glia to prevent its toxicity.

The impairment of cognitive and motor functions in humans and animals caused by methamphetamine (METH) administration underscores the importance of METH toxicity in cortical neurons. The heme oxygenase-1 (HO-1) exerts a cytoprotective effect against various neuronal injures; however, it remains unclear whether HO-1 is involved in METH-induced toxicity. We used primary cortical neuron/glia cocultures to explore the role of HO-1 in METH-induced toxicity. Exposure of cultured cells to various concentrations of METH (0.1, 0.5, 1, 3, 5, and 10 mM) led to cytotoxicity in a concentration-dependent manner. A METH concentration of 5 mM, which caused 50% of neuronal death and glial activation, was chosen for subsequent experiments. RT-PCR and Western blot analysis revealed that METH significantly induced HO-1 mRNA and protein expression, both preceded cell death. Double and triple immunofluorescence staining further identified HO-1-positive cells as activated astrocytes, microglia, and viable neurons, but not dying neurons. Inhibition of the p38 mitogen-activated protein kinase pathway significantly blocked HO-1 induction by METH and aggravated METH neurotoxicity. Inhibition of HO activity using tin protoporphyrine IX significantly reduced HO activity and exacerbated METH neurotoxicity. However, prior induction of HO-1 using cobalt protoporphyrine IX partially protected neurons from METH toxicity. Taken together, our results suggest that induction of HO-1 by METH via the p38 signaling pathway may be protective, albeit insufficient to completely protect cortical neurons from METH toxicity.

Neonatal status epilepticus alters prefrontal-striatal circuitry and enhances methamphetamine-induced behavioral sensitization in adolescence.

Neonatal seizures may alter the developing neurocircuitry and cause behavioral abnormalities in adulthood. We found that rats previously subjected to lithium-pilocarpine (LiPC)-induced neonatal status epilepticus (NeoSE) exhibited enhanced behavioral sensitization to methamphetamine (MA) in adolescence. Neurochemically, dopamine (DA) and metabolites were markedly decreased in prefrontal cortex (PFC) and insignificantly changed in striatum by NeoSE, but were increased in both PFC and striatum by NeoSE+MA. Glutamate levels were increased in both PFC and striatum in the NeoSE+MA group. DA turnover, an index of utilization and activity, was increased by NeoSE but reversed by MA in PFC. Gene expression of the regulator of G-protein signaling 4 (RGS4) was downregulated in PFC and striatum by NeoSE and further suppressed by MA. These findings suggest NeoSE affects both dopaminergic and glutamatergic systems in the prefrontal-striatal circuitry that manifests as enhanced behavioral sensitization to MA in adolescence.

Lovastatin improves histological and functional outcomes and reduces inflammation after experimental traumatic brain injury.

Traumatic brain injury (TBI) triggers a complex sequence of inflammatory responses that contribute to secondary injury. Statins have demonstrated neuroprotective effects against brain injury, but the underlying mechanisms remain unclear. This study evaluated the effects of lovastatin on a rat model of controlled cortical impact (CCI) injury. Our two hypotheses were that pre-administration of lovastatin would reduce functional deficits and extent of anatomical brain damage and that lovastatin would attenuate levels of pro-inflammatory cytokines. Rats were injected with lovastatin (4 mg/kg) or vehicle for 5 days and subjected to CCI. Neurological status was evaluated using rotarod and adhesive removal tests. Contusion volume and neuronal degeneration were examined using cresyl violet and FluoroJade B (FJB) histochemistry. Levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) mRNA and protein were assessed by real-time quantitative reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. Lovastatin significantly improved performance on both the rotarod and adhesive removal tests before post-injury day 7. Lovastatin also significantly reduced contusion volume (20%) and number of FJB-positive degenerating neurons (35%) at 4 days. These changes were associated with a significant decrease in levels of TNF-alpha and IL-1beta mRNA and protein at the contusion site at 6 h and 4 days, respectively. Our results show that pre-administration of lovastatin improved functional outcomes and reduced extent of brain damage, with a concomitant decrease in tissue levels of TNF-alpha and IL-1beta mRNA and protein. These findings suggest that lovastatin's protective mechanisms may be partly attributed to a dampening of the inflammatory response.

Rapid glia expression and release of proinflammatory cytokines in experimental Klebsiella pneumoniae meningoencephalitis.

The host immune/inflammatory response following CNS infection by Klebsiella pneumoniae remains poorly understood. Using a rat model of K. pneumoniae meningoencephalitis, we investigated the temporal profiles of brain proinflammatory cytokines and their cellular sources. Leukocyte counts significantly increased in cerebrospinal fluid (CSF) at 2 h after K. pneumoniae inoculation into the rat brain but were still much lower than blood leukocyte counts. However, concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in CSF were much higher than the simultaneously collected serum levels. The rapid increase in brain expression of these cytokines at the messenger RNA (mRNA) and protein levels occurred earlier than the onset of leukocytosis. Double immunofluorescence staining revealed the presence of TNF-alpha, IL-1beta, and IL-6 in astrocytes and microglia. Exposure of primary culture of glial cells to K. pneumoniae also resulted in time-dependent increases in the concentration of these cytokines in the culture media. Taken together, our results suggest that glial cells are an important early source of proinflammatory cytokines during K. pneumonia infection of CNS.

Dual effects of antioxidants in neurodegeneration: direct neuroprotection against oxidative stress and indirect protection via suppression of glia-mediated inflammation.

Oxidative stress, in which production of highly reactive oxygen species (ROS) and reactive nitrogen species (RNS) overwhelms antioxidant defenses, is a feature of many neurological diseases and neurodegeneration. ROS and RNS generated extracellularly and intracellularly by various processes initiate and promote neurodegeneration in CNS. ROS and RNS can directly oxidize and damage macromolecules such as DNA, proteins, and lipids, culminating in neurodegeneration in the CNS. Neurons are most susceptible to direct oxidative injury by ROS and RNS. ROS and RNS can also indirectly contribute to tissue damage by activating a number of cellular pathways resulting in the expression of stress-sensitive genes and proteins to cause oxidative injury. Moreover, oxidative stress also activates mechanisms that result in a glia-mediated inflammation that also causes secondary neuronal damage. Associated with neuronal injuries caused by many CNS insults is an activation of glial cells (particularly astrocytes and microglia) at the sites of injury. Activated glial cells are thus histopathological hallmarks of neurodegenerative diseases. Even though direct contact of activated glia with neurons per se may not necessarily be toxic, the immune mediators (e.g. nitric oxide and reactive oxygen species, pro-inflammatory cytokines and chemokines) released by activated glial cells are currently considered to be candidate neurotoxins. Therefore, study of the protective role of antioxidant compounds on inhibition of the inflammatory response and correcting the fundamental oxidant/antioxidant imbalance in patients suffering from neurodegenerative diseases are important vistas for further research. The purpose of this review is to summarize the current evidence in support of this critical role played by oxidative stress of neuronal and glial origin in neurodegenerative diseases. The mechanistic basis of the neuroprotective activity of antioxidants does not only rely on the general free radical trapping or antioxidant activity per se in neurons, but also the suppression of genes induced by pro-inflammatory cytokines and other mediators released by glial cells. We propose that combinations of agents which act at sequential steps in the neurodegenerative process can produce additive neuroprotective effects. A cocktail of multiple antioxidants with anti-inflammatory agents may be more beneficial in the prevention of neurodegenerative disease. A clearer appreciation of the potential therapeutic utility of antioxidants would emerge only when the complexity of their effects on mechanisms that interact to determine the extent of oxidative damage in vivo are more fully defined and understood.