Sustained Na⁺/H⁺ exchanger activation promotes gliotransmitter release from reactive hippocampal astrocytes following oxygen-glucose deprivation

Hypoxia ischemia (HI)-related brain injury is the major cause of long-term morbidity in neonates. One characteristic hallmark of neonatal HI is the development of reactive astrogliosis in the hippocampus. However, the impact of reactive astrogliosis in hippocampal damage after neonatal HI is not fully understood. In the current study, we investigated the role of Na +/H+ exchanger isoform 1 (NHE1) protein in mouse reactive hippocampal astrocyte function in an in vitro ischemia model (oxygen/glucose deprivation and reoxygenation, OGD/REOX). 2 h OGD significantly increased NHE1 protein expression and NHE1-mediated H+ efflux in hippocampal astrocytes. NHE1 activity remained stimulated during 1-5 h REOX and returned to the basal level at 24 h REOX. NHE1 activation in hippocampal astrocytes resulted in intracellular Na+ and Ca2+ overload. The latter was mediated by reversal of Na+/Ca2+ exchange. Hippocampal astrocytes also exhibited a robust release of gliotransmitters (glutamate and pro-inflammatory cytokines IL-6 and TNFα) during 1-24 h REOX. Interestingly, inhibition of NHE1 activity with its potent inhibitor HOE 642 not only reduced Na+ overload but also gliotransmitter release from hippocampal astrocytes. The noncompetitive excitatory amino acid transporter inhibitor TBOA showed a similar effect on blocking the glutamate release. Taken together, we concluded that NHE1 plays an essential role in maintaining H + homeostasis in hippocampal astrocytes. Over-stimulation of NHE1 activity following in vitro ischemia disrupts Na+ and Ca2+ homeostasis, which reduces Na+-dependent glutamate uptake and promotes release of glutamate and cytokines from reactive astrocytes. Therefore, blocking sustained NHE1 activation in reactive astrocytes may provide neuroprotection following HI. © 2014 Cengiz et al

Microcystic/Reticular Schwannoma of the Frontal Lobe: An Unusual Occurrence

Schwannoma is a benign peripheral nerve sheath tumor that typically involves cranial nerves of the head and neck region. Intraparenchymal occurrence of this tumor is uncommon. Even rarer in this site is the microcystic/reticular pattern of schwannoma. This histologic variant, first described in 2008, has a predilection for visceral organs. Herein, we report the first case of microcystic/reticular schwannoma of the frontal lobe

Inhibition of Na+/H+ Exchanger Isoform 1 Is Neuroprotective in Neonatal Hypoxic Ischemic Brain Injury

We investigated the role of Na+/H+ exchanger isoform 1 (NHE-1) in neonatal hypoxia/ischemia (HI). HI was induced by unilateral ligation of the left common carotid artery in postnatal day 9 (P9) mice, and subsequent exposure of animals to 8% O2 for 55 min. A pre/posttreatment group received a selective and potent NHE-1 inhibitor HOE 642 (0.5 mg/kg, intraperitoneally) 5 min before HI, then at 24 and 48 h after HI. A posttreatment group received HOE 642 (0.5 mg/kg) at 10 min, 24 h, and 48 h after HI. Saline injections were used as vehicle controls. The vehicle-control brains at 72 h after HI exhibited neuronal degeneration in the ipsilateral hippocampus, striatum, and thalamus, as identified with Fluoro-Jade C positive staining and loss of microtubule-associated protein 2 (MAP2) expression. NHE-1 protein was upregulated in glial fibrillary acidic protein–positive reactive astrocytes. In HOE 642–treated brains, the morphologic hippocampal structures were better preserved and displayed less neurodegeneration and a higher level of MAP2 expression. Motor-learning deficit was detected at 4 weeks of age after HI in the vehicle control group. Inhibition of NHE-1 in P9 mice not only reduced neurodegeneration during the acute stage of HI but also improved the striatum-dependent motor learning and spatial learning at 8 weeks of age after HI. These findings suggest that NHE-1–mediated disruption of ionic homeostasis contributes to striatal and CA1 pyramidal neuronal injury after neonatal HI. Antioxid. Redox Signal. 14, 1803–1813

Chronic Neurological Deficits in Mice after Perinatal Hypoxia and Ischemia Correlate with Hemispheric Tissue Loss and White Matter Injury Detected by MRI

We investigated the effects of perinatal hypoxia-ischemia (HI) on brain injury and neurological functional outcome at postnatal day (P) 30 through P90. HI was induced by exposing P9 mice to 8% O-2 for 55 min using the Vannucci HI model. Following HI, mice were treated with either vehicle control or Na+/H+ exchanger isoform 1 (NHE1) inhibitor HOE 642. The animals were examined by the accelerating rotarod test at P30 and the Morris water maze (MWM) test at P60. T-2-weighted MRI was conducted at P90. Diffusion tensor imaging (DTI) was subsequently performed in ex vivo brains, followed by immunohistochemical staining for changes in myelin basic protein (MBP) and neurofilament protein expression in the corpus callosum (CC). Animals at P30 after HI showed deficits in motor and spatial learning. T-2 MRI detected a wide spectrum of brain injury in these animals. A positive linear correlation was observed between learning deficits and the degree of tissue loss in the ipsilateral hemisphere and hippocampus. Additionally, CC DTI fractional anisotropy (FA) values correlated with MBP expression. Both FA and MBP values correlated with performance on the MWM test. HOE 642-treated mice exhibited improved spatial learning and memory, and less white matter injury in the CC. These findings suggest that HI-induced cerebral atrophy and CC injury contribute to the development of deficits in learning and memory, and that inhibition of NHE1 is neuroprotective in part by reducing white matter injury. T-2-weighted MRI and DTI are useful indicators of functional outcome after perinatal HI. Copyright (C) 2011 S. Karger AG, Base

Glutamate and pro-inflammatory cytokine release from hippocampal astrocytes following OGD/REOX.

<p><b>A.</b> Glutamate release in hippocampal astrocyte cultures was determined at 2(1 µM) or TBOA (100 µM) was present during REOX only. Data are mean ± SEM. N of 4 cultures were for all groups except for normoxia (n = 5). *p<0.05 vs. normoxic control, # p<0.05 vs. corresponding untreated. ND: not detectable. <b>Inset:</b> The reversal potential for the glutamate transport was plotted as a function of [Na⁺]_i. The known values for [Na]_o, [H]_o, [H]_i and [K]_o at baselines were used along with an assumed value for [Glu]_o of 0.01 µM, [Glu]_i of 5 mM and [K]_i of 70 mM. <b>B–D</b>. Release of innate immune cytokines in the culture medium of hippocampal astrocytes. HOE 642 (1 µM) was present during normoxia or REOX treatment. IL-1β (<b>B</b>) IL-6 (<b>C</b>), or TNF-α (<b>D</b>) were normalized to cell lysate protein and expressed as pg/mg protein. Data are mean ± SEM (n = 3). ND: not detectable. *p<0.05 vs. normoxic control. # p<0.05 vs. corresponding untreated.</p

Proposed role of NHE1 in reactive hippocampal astrocytes following OGD/REOX.

<p>In resting astrocytes under normoxic conditions, NHE1 and NCX function in removal of intracellular H⁺ and Ca²⁺. Glutamate transporters (EAAT) uptake glutamate (Glu) into astrocytes to maintain its low extracellular content. In response to OGD/REOX, up-regulation of NHE1 promotes sustained H⁺ efflux in exchange of Na⁺ influx in reactive astrocytes. This Na⁺ overload triggers the reversal mode operation of NCX and Ca²⁺ influx. The intracellular Ca²⁺ rise serves as a signal to release innate cytokines, while elevation of Na⁺_i leads to Glu release either via decreased uptake of Glu through EEAT or their reversed operation.</p

Pro-inflammatory cytokine release from LME-treated hippocampal astrocyte cultures following OGD/REOX.

<p>Release of innate immune cytokines was detected in the culture medium of hippocampal astrocytes at 2(1 µM) was present during normoxia or REOX treatment (with 5 mM LME). IL-1β (<b>A</b>), IL-6 (<b>B</b>), TNF-α (<b>C</b>). Cytokine content was normalized to cell lysate protein and expressed as pg/mg protein. Data are mean ± SEM (n = 3). *p<0.05 vs. normoxic control. # p<0.05 vs. corresponding untreated, ND: not detectable.</p

Up-regulation of NHE1 protein expression in reactive hippocampal astrocytes.

<p><b>A.</b> Expression of NHE1 protein in hippocampal astrocytes. Immunofluorescence signals of NHE1 and GFAP were shown under normoxia control or OGD (2 h) plus 0 h, 5 h, or 24 h REOX. <i>Green</i>: GFAP, <i>red</i>: NHE1 (arrows), <i>blue</i>: To-pro-3 nuclear staining. Scale Bar: 20 µm. <b>B, C.</b> Quantification of GFAP (<b>B</b>) and NHE1 protein (<b>C</b>) expression with immunoblotting. Hippocampal astrocytes were subjected to 2 h OGD followed by 5 or 24 h REOX. Total pixel density in each protein band (minus background) was calculated using Image J. Data are expressed as the ratio of either GFAP or NHE1 to the corresponding α-tubulin band and normalized to the control. Data are mean ± SEM (n = 4). *p<0.05 vs. normoxia.</p