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ABSTRACT: BACKGROUND: Hypoxia induces microglial activation which causes damage to the developing brain. Microglia derived inflammatory mediators may contribute to this process. Toll-like receptor 4 (TLR4) has been reported to induce microglial activation and cytokines production in brain injuries; however, its role in hypoxic injury remains uncertain. We investigate here TLR4 expression and its roles in neuroinflammation in neonatal rats following hypoxic injury. METHODS: One day old Wistar rats were subjected to hypoxia for 2 h. Primary cultured microglia and BV-2 cells were subjected to hypoxia for different durations. TLR4 expression in microglia was determined by RT-PCR, western blot and immunofluorescence staining. Small interfering RNA (siRNA) transfection and antibody neutralization were employed to downregulate TLR4 in BV-2 and primary culture. mRNA and protein expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta) and inducible nitric oxide synthase (iNOS) was assessed. Reactive oxygen species (ROS), nitric oxide (NO) and NF-kappaB levels were determined by flow cytometry, colorimetric and ELISA assays respectively. Hypoxia-inducible factor-1 alpha (HIF-1alpha) mRNA and protein expression was quantified and where necessary, the protein expression was depleted by antibody neutralization. In vivo inhibition of TLR4 with CLI-095 injection was carried out followed by investigation of inflammatory mediators expression via double immunofluorescence staining. RESULTS: TLR4 immunofluorescence and protein expression in the corpus callosum and cerebellum in neonatal microglia were markedly enhanced post-hypoxia. In vitro, TLR4 protein expression was significantly increased in both primary microglia and BV-2 cells post-hypoxia. TLR4 neutralization in primary cultured microglia attenuated the hypoxia-induced expression of TNF-alpha, IL-1beta and iNOS. siRNA knockdown of TLR4 reduced hypoxia-induced upregulation of TNF-alpha, IL-1beta, iNOS, ROS and NO in BV-2 cells. TLR4 downregulation-mediated inhibition of inflammatory cytokines in primary microglia and BV-2 cells was accompanied by the suppression of NF-kappaB activation. Furthermore, HIF-1alpha antibody neutralization attenuated the increase of TLR4 expression in hypoxic BV-2 cells. TLR4 inhibition in vivo attenuated the immunoexpression of TNF-alpha, IL-1beta and iNOS on microglia post-hypoxia. CONCLUSION: Activated microglia TLR4 expression mediated neuroinflammation via a NF-kappaB signaling pathway in response to hypoxia. Hence, microglia TLR4 presents as a potential therapeutic target for neonatal hypoxia brain injuries.
Journal of Neuroinflammation 02/2013; 10(1):23. · 3.83 Impact Factor
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ABSTRACT: The developing cerebellum is extremely vulnerable to hypoxia which can damage the Purkinje neurons. We hypothesized that this might be mediated by tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) derived from activated microglia as in other brain areas. One-day-old rats were subjected to hypoxia following, which the expression changes of various proteins in the cerebellum including hypoxia inducible factor-1α, TNF-α, IL-1β, TNF-R(1) and IL-1R(1) were analyzed. Following hypoxic exposure, TNF-α and IL-1β immunoexpression in microglia was enhanced coupled by that of TNF-R(1) and IL-1R(1) in the Purkinje neurons. Along with this, hypoxic microglia in vitro showed enhanced release of TNF-α and IL-1β whose receptor expression was concomitantly increased in the Purkinje neurons. In addition, nitric oxide (NO) level was significantly increased in the cerebellum and cultured microglia subjected to hypoxic exposure. Moreover, cultured Purkinje neurons treated with conditioned medium derived from hypoxic microglia underwent apoptosis but the incidence was significantly reduced when the cells were treated with the same medium that was neutralized with TNF-α/IL-1β antibody. We conclude that hypoxic microglia in the neonatal cerebellum produce increased amounts of NO, TNF-α and IL-1β which when acting via their respective receptors could induce Purkinje neuron death.
Brain Structure and Function 12/2012; · 5.63 Impact Factor
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ABSTRACT: The purpose of this study was to determine whether melatonin treatment would mitigate retinal ganglion cell (RGC) death in the developing retina following a hypoxic insult. Lipid peroxidation (LPO), glutathione (GSH), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) concentrations, expression of vascular endothelial growth factor receptors, Flt-1 and Flk-1, release of cytochrome c from mitochondria, and caspase-3 expression were examined in the retinas of 1-day-old rats at 3 hr to 14 days after a hypoxic exposure. The mRNA and protein expression of Flt-1 and Flk-1 and the tissue concentration of LPO, TNF-α, and IL-1β were upregulated significantly after the hypoxic exposure, whereas the content of GSH was decreased significantly. RGC cultures also showed increased LPO and decreased GSH levels after hypoxic exposure but these effects were reversed in cells treated with melatonin. TNF-α and IL-1β expression was specifically located on microglial cells, whereas Flt-1 and Flk-1 was limited to RGCs as confirmed by double immunofluorescence labeling. Cultures of hypoxic microglial cells treated with melatonin showed a significant reduction in the release of these cytokines as compared to untreated hypoxic cells. Hypoxia induced increase in the cytosolic cytochrome c and caspase-3 in RGCs was attenuated with melatonin treatment. The results suggest that, in hypoxic injuries, melatonin is neuroprotective to RGCs in the developing retina through its antioxidative, anti-inflammatory, and anti-apoptotic effects. Melatonin suppressed Flt-1 and Flk-1 expression in retinal blood vessels, which may result in reduced retinal vascular permeability and it also preserved mitochondrial function as shown by a reduction in cytochrome c leakage into the cytosol. The results may have therapeutic implications for the management of retinopathy of prematurity.
Journal of Pineal Research 09/2012; · 5.79 Impact Factor
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ABSTRACT: It is well established that hypoxia causes excess accumulation of glutamate in developing neural tissues. This study aimed to elucidate the mechanism by which glutamate can cause retinal ganglion cell (RGC) death through the N-methyl-D-aspartate (NMDA) receptors (NR) in the developing retina. One-day-old Wistar rats were exposed to hypoxia for 2 hours and then killed at different time points. Normal age-matched rats were used as controls. NR1, NR2A-D, and NR3A messenger RNA and protein expression showed significant increases over control values, notably at early time points (3 hours to 7 days) after the hypoxic exposure, and immunoexpression of NR1, NR2A-D and NR3A on retinal ganglion cells (RGCs) was enhanced in hypoxic rats and this was confirmed in cultured hypoxic RGCs. Ca(2+) influx in cultured RGCs was increased after hypoxic exposure, and the intracellular Ca(2+) concentration was suppressed by MK-801. Mitochondrial permeability transition pore opening, mitochondrial/cytosolic cytochrome c, and cytosolic caspase-3 expression levels were significantly increased in the hypoxic RGCs. These increases were reversed by MK-801, suggesting that the NMDA receptor subunits in the retina respond rapidly to the hypoxia-induced glutamate overload that leads to the cascade of events that result in RGC death.
Journal of Neuropathology and Experimental Neurology 04/2012; 71(4):330-47. · 4.26 Impact Factor
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ABSTRACT: To determine the effects on the electroretinogram (ERG) of retinal capillary closure induced in the pig by embolization with microspheres.
Fourteen Yorkshine Landrace pigs of 25- to 45-kg body weight were used. With a customized cannula introduced into the external carotid artery, 10-μm diameter microspheres were delivered to the origin of the vessel that supplies blood to the eye in the pig. Fundus fluorescein angiography and electroretinography were performed between days 7 and 28 post injection. The ERG responses of embolized eyes were compared with those of the contralateral nonembolized eyes.
The amplitudes of the scotopic b-wave (P = 0.002), the maximal b-wave (P < 0.010), the photopic a-wave (P < 0.001) and b-wave (P < 0.001), and the scotopic oscillatory potentials (OPs) (P = 0.025) and photopic OPs (P = 0.036) were significantly reduced in embolized eyes. The reduction of these ERG amplitudes was significantly correlated with the number of microspheres in the retina. There was no significant difference in the combined rod-cone bright flash (maximal) ERG a-wave amplitude between eyes with and without microspheres. Implicit times, however, were similar in embolized and control eyes.
In eyes embolized with microspheres, the amplitudes of most ERG components were significantly reduced without alteration of their implicit times. The magnitude of ERG amplitude reduction correlated with the number of microspheres in the retina.
Investigative ophthalmology & visual science 03/2012; 53(4):2218-25. · 3.43 Impact Factor
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ABSTRACT: Amoeboid microglial cells (AMCs) in the developing brain display surface receptors and antigens shared by the monocyte-derived tissue macrophages. Activation of AMCs in the perinatal brain has been associated with periventricular white matter damage in hypoxic-ischemic conditions. The periventricular white matter, where the AMCs preponderate, is selectively vulnerable to hypoxia as manifested by death of premyelinating oligodendrocytes and degeneration of axons leading to neonatal mortality and long-term neurodevelopmental deficits. AMCs respond vigorously to hypoxia by producing excess amounts of inflammatory cytokines e.g. the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) along with glutamate, nitric oxide (NO) and reactive oxygen species which collectively cause oligodendrocyte death, axonal degeneration as well as disruption of the immature blood brain barrier. A similar phenomenon is observed in the hypoxic developing cerebellum in which activated AMCs induced Purkinje neuronal death through production of TNF-α and IL-1β via their respective receptors. Hypoxia is also implicated in retinopathy of prematurity in which activation of AMCs has been shown to cause retinal ganglion cell death through production of TNF-α and IL-1β and NO. Because AMCs play a pivotal role in hypoxic injuries in the developing brain affecting both neurons and oligodendrocytes, a fuller understanding of the underlying molecular mechanisms of microglial activation under such conditions would be desirable for designing of a novel therapeutic strategy for management of hypoxic damage.
Journal of Neuroimmune Pharmacology 02/2012; · 4.57 Impact Factor
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ABSTRACT: The review aims to elucidate the potential of microglia as a therapeutic target in alleviating Alzheimer's Disease (AD). Microglia are the resident immune cells in the brain which respond to the presence of the hallmarks of AD, amyloid-beta (A beta) plaques and neurofibrillary tangles (NFT). Activated microglia are able to phagocytose and secrete pro-inflammatory and anti-inflammatory cytokines. However, the eventual accumulation of excess A beta peptides and NFT in AD means that microglial clearance of pathogens has been impaired. Pro-inflammatory cytokines may also contribute to the neurodegeneration. Based on the amyloid cascade hypothesis, A beta-activated microglia can produce pro-inflammatory cytokines which may exacerbate the hyperphosporylation of tau proteins that forms NFT in AD pathology. Microglial activation can thus be manipulated to prevent neurodegeneration and promote neuroprotection through several therapeutic agents and methods. Further studies regarding comprehensive microglial response towards A beta and NFT are required to develop an effective treatment of AD involving microglia.
Frontiers in bioscience (Scholar edition) 01/2012; 4:1402-12.
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ABSTRACT: This study was aimed to examine the role of iron in causing periventricular white matter (PWM) damage following a hypoxic injury in the developing brain. Along with iron, the expression of iron regulatory proteins (IRPs) and transferrin receptor (TfR), which are involved in iron acquisition, was also examined in the PWM by subjecting 1-d-old Wistar rats to hypoxia. Apart from an increase in iron levels in PWM, Perls' iron staining showed an increase of intracellular iron in the preponderant amoeboid microglial cells (AMCs) in the tissue. In response to hypoxia, the protein levels of IRP1, IRP2, and TfR in PWM and AMCs were significantly increased. In primary microglial cultures, administration of iron chelator deferoxamine reduced the generation of iron-induced reactive oxygen and nitrogen species and proinflammatory cytokines such as tumor necrosis factor-α and interleukin-1β. Primary oligodendrocytes treated with conditioned medium from hypoxic microglia exhibited reduced glutathione levels, increased lipid peroxidation, upregulated caspase-3 expression, and reduced proliferation. This was reversed to control levels on treatment with conditioned medium from deferoxamine treated hypoxic microglia; also, there was reduction in apoptosis of oligodendrocytes. The present results suggest that excess iron derived primarily from AMCs might be a mediator of oligodendrocyte cell death in PWM following hypoxia in the neonatal brain.
Journal of Neuroscience 12/2011; 31(49):17982-95. · 7.11 Impact Factor
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Charanjit Kaur
Recent Patents on Endocrine Metabolic & Immune Drug Discovery 05/2011; 5(2):78-9.
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ABSTRACT: The present study was focused on identifying the expression of N-methyl D-aspartate receptor (NMDAR) subunits on activated microglia and to determine their role in the pathogenesis of periventricular white matter damage (PWMD) in neonatal rats following hypoxia. One day old wistar rats were subjected to hypoxia (5% O(2) ; 95% N(2) ) and the mRNA and protein expression of NMDAR subunits (NR1, NR2A-D, and NR3A) in the periventricular white matter (PWM) was determined at different time points (3,24 h, 3, 7, and 14 days) following hypoxic exposure. Immunoexpression of NR1 and NR2A-D was localized in amoeboid microglial cells (AMC) suggesting the presence of functional NMDARs in them. The expression of NMDAR in primary microglial cultures was ascertained by RT-PCR analysis and double immunofluorescence studies. The functionality of the microglial NMDAR in cultured microglial cells was examined by monitoring calcium movements in cells with fura-2. In primary microglial cultures, hypoxia induced the nuclear translocation of NF-κB which was suppressed by administration of MK801, an NMDAR antagonist. MK801 also down regulated the hypoxia-induced expression of tumor necrosis factor-α, interleukin-1β, inducible nitric oxide synthase (iNOS), and nitric oxide (NO) production by microglia which may be mediated by the NF-κB signaling pathway. NO produced by microglia is known to cause death of oligodendrocytes in the developing PWM. In this connection, pharmacological agents such as MK801, BAY (NF-κB inhibitor), and 1400w (iNOS inhibitor) proved to be beneficial since they reduced the hypoxia-induced iNOS expression, NO production, and a corresponding reduction in the death of oligodendrocytes following hypoxia.
Glia 04/2011; 59(4):521-39. · 4.82 Impact Factor
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ABSTRACT: Major changes in arterial pressure, autonomic, and respiratory activity occur in response to hypoxia. We analyzed structural damage and increased vascular permeability in the ventrolateral medulla and nucleus tractus solitarius, which control autonomic, respiratory, and cardiovascular functions in adult Wistar rats subjected to 2 hours of hypoxia (7% oxygen + 93% nitrogen) for up to 14 days after hypoxicexposure. Brainstem tissue levels of vascular endothelial growth factor (VEGF), nitric oxide (NO), and glutamate were significantly increased over control levels after hypoxic injury. By electron microscopy, swollen neurons and dendrites, degenerating axons, disrupted myelin sheaths, and swollen astrocyte processes were observed in the nucleus tractus solitarius and ventrolateral medulla. Leakage of intravenously administered horseradish peroxidase was observed through vascular walls in hypoxic rats. These results suggest that increased VEGF and NO production in hypoxia resulted in increased vascular permeability, which, along with increased levels of glutamate, may have induced structural alterations of the neurons, dendrites, and axons. Administration of the antioxidant neurohormone melatonin (10mg/kg) before and after the hypoxia reduced VEGF, NO, and glutamate levels and improved ultrastructural abnormalities induced by hypoxia exposure, suggesting that it may have a therapeutic potential in reducing hypoxia-associated brainstem damage.
Journal of Neuropathology and Experimental Neurology 03/2011; 70(3):201-17. · 4.26 Impact Factor
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ABSTRACT: Sphingosine kinase 1 (SphK1), a key enzyme responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P) has been shown to be expressed in monocytes and monocyte-derived peripheral macrophages. This study demonstrates SphK1 immunoexpression in amoeboid microglial cells (AMC), a nascent monocyte-derived brain macrophage in the corpus callosum of developing rat brain. SphK1 immunofluorescence expression, which appeared to be weak in AMC in normal brain, was markedly induced by lipopolysaccharide (LPS) or hypoxia treatment. Western blot analysis also showed increased expression level of SphK1 in the corpus callosum rich in AMC after LPS treatment. Detection of SphK1 mRNA and its upregulation after LPS treatment was confirmed in primary culture AMC by RT-PCR. Administration of N, N-dimethylsphingosine (DMS), a specific inhibitor of SphK1, effectively reduced upregulated SphK1 immunoexpression in AMC both in vivo and in vitro. This was corroborated by western blot which showed a decrease in SphK1 protein level of callosal tissue with DMS pretreatment. Remarkably, LPS-induced upregulation of the transcription factor NFκB was suppressed by DMS. We conclude that SphK1 expression in AMC may be linked to regulation of proinflammatory cytokines via an NFκB signaling pathway.
Journal of Neuroinflammation 02/2011; 8:13. · 3.83 Impact Factor
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ABSTRACT: Hypoxic injury, including that resulting in the retinopathy of prematurity, may induce retinal ganglion cell (RGC) death in the neonatal retina. We hypothesized that this may be mediated by excess production of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) by microglia. One-day-old Wistar rats were subjected to hypoxia for 2 h and the expression of TNF-α and IL-1β and their receptors was determined in the retina. The mRNA and protein expression of TNF-α, IL-1β, TNF-receptor 1 (TNF-R(1)), and IL-1 receptor 1 (IL-1R(1)) and the tissue concentration of TNF-α and IL-1β were up-regulated significantly after the hypoxic exposure. TNF-α and IL-1β immunoreactivity was localized in microglial cells, whereas that of TNF-R(1) and IL-1R(1) was restricted to RGCs, as confirmed by double immunofluorescence labelling. Along with this, increased expression of monocyte chemoattractant protein-1 and its receptor CCR2 was detected in the microglia. Primary cultured microglia subjected to hypoxia showed enhanced release of TNF-α and IL-1β. Primary cultured retinal ganglion cells (RGCs) treated with conditioned medium derived from hypoxic microglia showed enhanced apoptosis, which was significantly reduced when the cells were treated with microglia conditioned medium neutralized with TNF-α/IL-1β antibody. Our results suggest that activated microglial cells in hypoxic neonatal retina produce increased amounts of TNF-α and IL-1β that could induce RGC death.
The Journal of Pathology 01/2011; 224(2):245-60. · 6.32 Impact Factor
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ABSTRACT: The developing brain is susceptible to hypoxic damage because of its high oxygen and energy requirements. Hypoxia-induced inflammatory response has been recognized as one of the main culprits in the development of hypoxic brain injury. In this regard, a hallmark feature is microglial activation which results in overproduction of inflammatory cytokines, free radicals and nitric oxide. Concomitantly, activated microglia exhibit enhanced expression of ion channels such as Kv1.2, Kv1.1 and Nav which further promote the release of inflammatory cytokines, chemokines and reactive oxygen species. Through the above-mentioned inflammatory mediators, activated microglia induce neuronal loss, axonal damage and oligodendroglial death along with myelination disturbances. Our recent studies have extended that tumor necrosis factor-alpha, interleukin-1beta, monocyte chemoattractant protein-1 and macrophage colony stimulating factor produced by activated microglia are linked to the pathogenesis of periventricular white matter damage in the hypoxic brain. It is envisaged that a better understanding of the interactions between microglia and neurons, axons and oligodendrocytes is key to the development of effective preventive and therapeutic strategies for mitigation of hypoxic brain injury.
Frontiers in bioscience (Scholar edition) 01/2011; 3:884-900.
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ABSTRACT: To investigate the feasibility of creating an animal model of selective retinal capillary closure to mimic the capillary closure that occurs in diabetic retinopathy.
Fluorescent microspheres of 10- or 15-μm diameter were delivered to one eye of anesthetized pigs via a customized cannula advanced through the carotid arterial system to the origin of the external ophthalmic artery that supplies blood to the eye in this species. After preliminary trials in 10 pigs, embolization was performed in one eye of 34 animals that were allowed to survive for 7, 14, or 28 days. Embolized eyes were assessed by fluorescein angiography, electroretinography (ERG), and, after enucleation, light (LM) and electron (EM) microscopy.
The microspheres were detectable in the retina immediately after embolization, were restricted to the nerve fiber layer of the retina, and remained thereafter within the retina for periods up to 28 days. They effectively occluded embolized capillaries and some precapillary arterioles. No systemic or cerebral adverse effects were noted, thus allowing survival and subsequent follow-up. Embolization caused a reduction in the b-wave amplitude and the oscillatory potentials of the rod-cone bright-flash ERG but did not affect the amplitude of the a-wave. Embolization induced extracellular and intracellular edema confined to the inner and mid retina, and as a result the retinas of embolized eyes were thicker than those of fellow, nonembolized eyes. The outer retina and RPE were unaffected.
This survival model of retinal embolization with microspheres should be useful in the study of the retinal effects of the capillary closure that may occur in diabetic eyes.
Investigative ophthalmology & visual science 12/2010; 51(12):6700-9. · 3.43 Impact Factor
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ABSTRACT: Inflammation in the periventricular white matter (PWM) of hypoxic neonatal brain causes myelination disturbances. In this connection, macrophage colony-stimulating factor (M-CSF) has been reported to regulate release of proinflammatory cytokines that may be linked to PWM damage. We sought to determine if M-CSF derived from amoeboid microglial cells (AMC) would promote proinflammatory cytokine production by astrocytes in the PWM following hypoxic exposure, and, if so, whether it is associated with axon degeneration and myelination disturbances. In 1-day hypoxic rats, expression of M-CSF was upregulated in AMC. This was coupled with increased expression of CSF-1 receptor, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in astrocytes, and TNF-receptor 1 and IL-receptor 1 on the axons. Neurofilament-200 immunopositive axons and myelin basic protein immunopositive processes appeared to undergo disruption in 14-days hypoxic rats. By electron microscopy, some axons showed degenerative changes affecting the microtubules and myelin sheath. Primary cultured microglial cells subjected to hypoxia showed enhanced release of M-CSF. Remarkably, primary cultured astrocytes treated with conditioned-medium derived from hypoxic microglia or M-CSF exhibited increased production of TNF-alpha and IL-1beta. Our results suggest that AMC-derived M-CSF promotes astrocytes to generate proinflammatory cytokines, which may be involved in axonal damage following a hypoxic insult.
Brain Pathology 09/2010; 20(5):909-25. · 3.99 Impact Factor
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ABSTRACT: Microglia express cyclooxygenase-2 (COX-2) and microsomal prostaglandin-E synthase (mPGES-1) but their localization in the amoeboid microglial cells (AMC), considered to be the nascent brain macrophages, in the developing brain has remained unexplored; furthermore, their interrelation and regulation have also remained to be fully elucidated. We show here that AMC in postnatal rat brain constitutively expressed COX-2 and mPGES-1 whose immunoexpression was upregulated in rats given lipopolysaccharide (LPS) injections. Reverse transcriptase-polymerase chain reaction and Western blot analysis of the callosal tissue rich in AMC revealed that COX-2 and mPGES-1 mRNA and protein expression was augmented following LPS injections. BV-2 cells also exhibited COX-2 and mPGES-1 expression which was enhanced by LPS. However, in cells treated with LPS coupled with COX-2 neutralization, the mRNA expression levels of COX-2, mPGES-1, tumor necrosis factor-alpha, interleukin-1beta and inducible nitric oxide synthase were significantly suppressed; production of prostaglandin E(2) and reactive oxygen species also decreased. Western blot analysis confirmed the changes of protein levels of the above mediators. Remarkably, COX-2 neutralization concomitantly suppressed the protein expression levels of nuclear factor-kappa B (NF-kappaB), phos-NF-kappaB and phos-IkappaB-alpha as well as translocation of NF-kappaB as determined by flow cytometry. In conclusion, AMC in the developing brain expressed COX-2 and mPGES-1 notably when stimulated by LPS. It is suggested that this may be involved in local inflammation during development. Our results have further shown that COX-2 neutralization may be effective in suppressing production of inflammatory mediators and hence its potential use in alleviating neuroinflammation.
Journal of Neuroscience Research 12/2009; 88(7):1577-94. · 2.74 Impact Factor
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ABSTRACT: This study investigated the glutamate concentration and cellular localization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid glutamate receptors (AMPA GluR2, GluR3, GluR4) along with insulin-like growth factors (IGF)-1 and -2 expression in the periventricular white matter (PWM) of neonatal rats with the aim to determine their involvement in PWM injury in hypoxia. In response to hypoxia, the PWM tissue concentration of glutamate and IGF-1 as well as mRNA and protein expression of GluR2, GluR3, GluR4, IGF-1, and -2 was upregulated. Immunoexpression of GluR2/3 and GluR4 were localized in the amoeboid microglial cells (AMC) and oligodendrocytes while that of IGF-1 and -2 were confined to AMC. In primary microglial cultures subjected to hypoxia, administration of exogenous glutamate decreased IGF-1 but increased the release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) by the cells. Furthermore, silencing of the IGF-1 and -2 genes by RNA interference in primary microglial cultures and BV-2 cells downregulated the expression of these growth factors whereas production of glutamate, TNF-alpha, and IL-1beta in these cells was upregulated. It is suggested that increased IGF-1 and -2 expressions may be an early protective mechanism in attenuating the hypoxic damage in PWM but a subsequent glutamate-induced decrease of these growth factors may cause cellular injury due to excitotoxicity and increased production of inflammatory cytokines. In this connection, melatonin and edaravone were beneficial in enhancing IGF-1 and reducing glutamate release.
Glia 09/2009; 58(5):507-23. · 4.82 Impact Factor
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ABSTRACT: This study was undertaken to examine the effects of an acute hypoxic exposure on the retinal cells and production of vascular factors such as vascular endothelial growth factor (VEGF) and nitric oxide (NO), which may affect vascular permeability in the developing retina.
Retinas of 1-day-old rats were examined at 3 hours to 14 days after hypoxic exposure. The mRNA and protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha), VEGF, endothelial nitric oxide synthase (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) were determined by real-time RT-PCR, Western blot analysis, and immunohistochemistry. Electron microscopy was used to examine the structural alterations in retinal cells, and rhodamine isothiocyanate (RhIC) or horseradish peroxidase (HRP) was administered intraperitoneally or intravenously to determine vascular permeability.
The mRNA and protein expression of HIF-1alpha, VEGF, eNOS, nNOS, and iNOS, along with VEGF concentration and NO production, were increased in response to hypoxia. Swollen Müller cell processes, apoptotic and necrotic cells in the inner nuclear layer, and changes in ganglion cells such as swollen and disrupted mitochondria were observed in hypoxic animals. Increased leakage of RhIC and HRP from retinal and hyaloid vessels was seen after hypoxic exposure.
The authors suggest that increased VEGF and NO production in hypoxia resulted in increased vascular permeability, leading to changes in Müller cells and degeneration of neural cells. Melatonin administration reduced VEGF and NO production, diminished leakage of RhIC and HRP, and promoted cell proliferation, suggesting this as a potential therapeutic agent in reducing hypoxia-associated damage in the developing retina.
Investigative ophthalmology & visual science 06/2009; 50(11):5364-74. · 3.43 Impact Factor
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ABSTRACT: Retinal hypoxia is the potentially blinding mechanism underlying a number of sight-threatening disorders including central retinal artery occlusion, ischemic central retinal vein thrombosis, complications of diabetic eye disease and some types of glaucoma. Hypoxia is implicated in loss of retinal ganglion cells (RGCs) occurring in such conditions. RGC death occurs by apoptosis or necrosis. Hypoxia-ischemia induces the expression of hypoxia inducible factor-1alpha and its target genes such as vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS). Increased production of VEGF results in disruption of the blood retinal barrier leading to retinal edema. Enhanced expression of NOS results in increased production of nitric oxide which may be toxic to the cells resulting in their death. Excess glutamate release in hypoxic-ischemic conditions causes excitotoxic damage to the RGCs through activation of ionotropic and metabotropic glutamate receptors. Activation of glutamate receptors is thought to initiate damage in the retina by a cascade of biochemical effects such as neuronal NOS activation and increase in intracellular Ca(2+) which has been described as a major contributing factor to RGC loss. Excess production of proinflammatory cytokines also mediates cell damage. Besides the above, free-radicals generated in hypoxic-ischemic conditions result in RGC loss because of an imbalance between antioxidant- and oxidant-generating systems. Although many advances have been made in understanding the mediators and mechanisms of injury, strategies to improve the damage are lacking. Measures to prevent neuronal injury have to be developed.
Clinical ophthalmology (Auckland, N.Z.) 01/2009; 2(4):879-89.
