Viswanathan Sivakumar

Hypoxia‐induced astrocytic reaction and increased vascular permeability in the rat cerebellum

Hypoxia is an important factor linked to induction of vascular leakage and formation of brain edema. In this connection, astrocytes associated closely with the blood vessels are deemed to be involved. This study investigated the response of astrocytes to hypoxia in the adult rat cerebellum, and along with this, the integrity of the blood–brain barrier (BBB) was assessed using fluorescent and electron dense tracers. In rats subjected to hypoxia, mRNA and protein expression of hypoxia inducible factor‐1α (HIF‐1α), vascular endothelial growth factor (VEGF), glial fibrillary acidic protein (GFAP), and aquaporin‐4 (AQ4) was significantly increased. VEGF and AQ4 immunoreactive cells were identified as astrocytes by double immunofluorescence labeling. Increased VEGF tissue concentration and astrocytic swelling as observed in hypoxic rats were reduced after melatonin administration. Following intraperitoneal or intravenous injection of rhodamine isothiocyanate (RhIC) or horseradish peroxidase (HRP), leakage of both tracers was observed in hypoxic rats but not in the controls indicating that functional integrity of BBB is compromised in hypoxia/reoxygenation. Enhanced gene and protein expression of VEGF may contribute to increased permeability of blood vessels. AQ4, a water transporting protein, is upregulated in astrocytes in hypoxia suggesting the cells are involved in edema formation. To this end, melatonin may be beneficial in reducing edema as it reduced VEGF concentration and, hence, vascular permeability.

Amoeboid Microglia in the Periventricular White Matter Induce Oligodendrocyte Damage through Expression of Proinflammatory Cytokines via MAP Kinase Signaling Pathway in Hypoxic Neonatal Rats

Hypoxic injury in the perinatal period results in periventricular white matter (PWM) lesions with axonal damage and oligodendroglial loss. It also alters macrophage function by perpetuating expression of inflammatory mediators. Relevant to this is the preponderance of amoeboid microglial cells (AMC) characterized as active macrophages in the developing PWM. This study aimed to determine if AMC produce proinflammatory cytokines that may be linked to the oligodendroglial loss observed in hypoxic PWM damage (PWMD). Wistar rats (1 day old) were subjected to hypoxia, following which upregulated expression of tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), TNF receptor 1 (TNF‐R1) and IL‐1 receptor 1 (IL‐1R1) was observed. This was coupled with apoptosis and expression of TNF‐R1 and IL‐1R1 in oligodendrocytes. Primary cultured microglial cells subjected to hypoxia (3% oxygen, 5% CO2 and 92% nitrogen) showed enhanced expression of TNF‐α and IL‐1β. Furthermore, mitogen‐activated protein (MAP) kinase signaling pathway was involved in the expression of TNF‐α and IL‐1β in microglia subjected to hypoxia. Our results suggest that following a hypoxic insult, microglial cells in the neonatal rats produce inflammatory cytokines such as TNF‐α and IL‐1β via MAP kinase signaling pathway. These cytokines are detrimental to oligodendrocytes resulting in PWM lesion.

Hypoxic damage to the periventricular white matter in neonatal brain: role of vascular endothelial growth factor, nitric oxide and excitotoxicity.

The present study examined factors that may be involved in the development of hypoxic periventricular white matter damage in the neonatal brain. Wistar rats (1‐day old) were subjected to hypoxia and the periventricular white matter (corpus callosum) was examined for the mRNA and protein expression of hypoxia‐inducible factor‐1α (HIF‐1α), endothelial, neuronal and inducible nitric oxide synthase (eNOS, nNOS and iNOS), vascular endothelial growth factor (VEGF) and N‐methyl‐D‐aspartate receptor subunit 1 (NMDAR1) between 3 h and 14 days after hypoxic exposure by real‐time RT‐PCR, western blotting and immunohistochemistry. Up‐regulated mRNA and protein expression of HIF‐1α, VEGF, NMDAR1, eNOS, nNOS and iNOS in corpus callosum was observed in response to hypoxia. NMDAR1 and iNOS expression was found in the activated microglial cells, whereas VEGF was localized to astrocytes. An enzyme immunoassay showed that the VEGF concentration in corpus callosum was significantly higher up to 7 days after hypoxic exposure. NO levels, measured by colorimetric assay, were also significantly higher in hypoxic rats up to 14 days after hypoxic exposure as compared with the controls. A large number of axons undergoing degeneration were observed between 3 h and 7 days after the hypoxic exposure at electron‐microscopic level. Our findings point towards the involvement of excitotoxicity, VEGF and NO in periventricular white matter damage in response to hypoxia.

Blood-retinal barrier disruption and ultrastructural changes in the hypoxic retina in adult rats : the beneficial effect of melatonin administration

Reactive changes in astrocytes and Müller cells in the retina of adult rats subjected to hypoxia were investigated. Along with this, the integrity of the blood–retinal barrier (BRB) was assessed using fluorescent and electron‐dense tracers. In hypoxic rats, mRNA and protein expression of glial fibrillary acidic protein (GFAP) and aquaporin‐4 (AQ4) were significantly increased. AQ4 immunoreactive cells were identified as astrocytes and Müller cells by double immunofluorescence labelling. Another alteration in the hypoxic retina was marked reduction in melatonin content compared to controls. In this connection, administration of exogenous melatonin reduced the tissue concentration of vascular endothelial growth factor (VEGF) and nitric oxide (NO); both were elevated in hypoxic rats. A major structural change in the hypoxic retina was swelling of astrocyte and Müller cell processes but this was noticeably attenuated after melatonin administration. Following an intraperitoneal or intravenous injection of rhodamine isothiocyanate (RhIC) or horseradish peroxidase (HRP), leakage of both tracers was observed in the retina in hypoxic rats but not in the controls, indicating that the functional integrity of the BRB is compromised in hypoxia/reoxygenation. It is suggested that enhanced tissue concentration of VEGF and NO production in the hypoxic retina contribute to increased permeability of the retinal blood vessels. The concurrent up‐regulation of AQ4, a water‐transporting protein, in astrocytes and Müller cells in hypoxia suggests its involvement in oedema formation. Since melatonin effectively reduced the vascular permeability in the retina of hypoxic rats, as evidenced by reduced leakage of RhIC, we suggest that its administration may be of potential benefit in the management of retinal oedema associated with retinal hypoxia. Copyright

Retinal ganglion cell death is induced by microglia derived pro‐inflammatory cytokines in the hypoxic neonatal retina

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‐R1), and IL‐1 receptor 1 (IL‐1R1) 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‐R1 and IL‐1R1 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. Copyright

Expression of N-methyl D-aspartate receptor subunits in amoeboid microglia mediates production of nitric oxide via NF-κB signaling pathway and oligodendrocyte cell death in hypoxic postnatal rats.

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% O2; 95% N2) 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.

Melatonin protects periventricular white matter from damage due to hypoxia.

Abstract:  This study investigated the potential of melatonin in ameliorating hypoxic damage to the periventricular white matter (PWM) in the neonatal brain. Vascular endothelial growth factor (VEGF), nitric oxide (NO), glutathione (GSH) and malondialdehyde (MDA) content in the PWM of 1‐day‐old rats subjected to hypoxia for a period of 2 hr was examined. Vascular endothelial growth factor, NO and MDA concentration was increased whereas that of GSH was reduced after the hypoxic exposure. Additionally, degenerating axons, apoptotic and necrotic cells and vacuolation of capillary endothelial cells were observed in the PWM. The neighboring ependymal and choroid plexus cells also appeared to undergo structural alterations. Increased vascular permeability in the PWM of hypoxic rats was evidenced by the leakage of rhodamine isothiocyanate (RhIC) which was taken up by the amoeboid microglial cells. In vitro experiments showed increased apoptosis in OLN‐93 cells, an oligodendrocytic cell line, following hypoxic exposure. Hypoxic rats treated with melatonin showed reduced VEGF, NO and MDA concentrations, increased GSH content and reduced RhIC leakage in the PWM. The ultrastructure of axons, endothelial, ependymal and choroid plexus epithelial cells appeared relatively normal in the hypoxic animals treated with melatonin. The incidence of apoptotic OLN‐93 cells was also reduced with melatonin treatment. We suggest that the protective effects of melatonin on various parameters in the PWM of hypoxic neonatal brains were due to its antioxidant properties.

Expression of Notch-1 receptor and its ligands Jagged-1 and Delta-1 in amoeboid microglia in postnatal rat brain and murine BV-2 cells

Notch‐1 receptor signaling pathway is involved in neuronal and glial differentiation. Its involvement in microglial functions, however, has remained elusive. This study reports the localization of Notch‐1 receptor immunoreactivity in the amoeboid microglial cells (AMC) in the postnatal rat brain. By immunofluorescence, Notch‐1 receptor was colocalized with its ligands, Jagged‐1 and Delta‐1, in the AMC in the corpus callosum and subventricular zone. Notch‐1 immunopositive cells were confirmed to be microglia labeled by OX42 and lectin. Immunoexpression of Notch‐1 receptor was progressively reduced with age. Western blot analysis showed that Notch‐1 protein level in the corpus callosum in which the AMC were heavily populated was concomitantly decreased. In postnatal rats challenged with lipopolysaccharide (LPS), Notch‐1 receptor immunofluorescence in AMC was noticeably enhanced. Furthermore, Notch‐1 protein level in the corpus callosum was increased as revealed by Western blotting analysis. In primary microglial culture treated with LPS, mRNA expression of Notch‐1 and its ligand Jagged‐1 was upregulated but that of Delta‐1 was reduced. The expression pattern of Notch‐1 and its ligands was confirmed in murine BV‐2 cells. Furthermore, Notch‐1 neutralization with its antibody reduced its protein expression. More importantly, neutralization of Notch‐1 concomitantly suppressed the mRNA expression of IL‐6, IL‐1, M‐CSF, and iNOS; TNF‐α, mRNA expression, however, was enhanced. Western blot confirmed the changes of protein level of the above except for IL‐6, which remained relatively unaltered. It is concluded that Notch‐1 signaling in the AMC and LPS‐activated microglia/BV‐2 cells modulates the expression of proinflammatory cytokines and nitric oxide.

Cellular and Vascular Changes in the Retina of Neonatal Rats after an Acute Exposure to Hypoxia

PURPOSE 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Role of glutamate and its receptors and insulin-like growth factors in hypoxia induced periventricular white matter injury†

This study investigated the glutamate concentration and cellular localization of α‐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‐α (TNF‐α) and interleukin‐1β (IL‐1β) 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‐α, and IL‐1β 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.