Nicole M. Jones

Hypoxic Preconditioning Induces Changes in HIF-1 Target Genes in Neonatal Rat Brain:

Hypoxic preconditioning induces tolerance to hypoxic-ischemic injury in neonatal rat brain and is associated with changes in gene expression. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that is strongly induced by hypoxia or the hypoxia-mimetic compound cobalt chloride (CoCl2). Hypoxia-inducible factor-1 modulates the expression of several target genes including the glycolytic enzymes, glucose transporter-1 (GLUT-1), and erythropoietin. Recently, HIF-1 expression was shown to increase after hypoxic and CoCl2 preconditioning in newborn rat brain. To study the involvement of HIF-1 target genes in neonatal hypoxia-induced ischemic tolerance, the authors examined the brains of newborn rats after exposure to hypoxia (8% O2 for 3 hours) or injection of CoCl2 (60 mg/kg). Preconditioning with hypoxia or CoCl2 24 hours before hypoxia-ischemia afforded a 96% and 76% brain protection, respectively, compared with littermate control animals. Hypoxic preconditioning increased the expression of GLUT-1 mRNA and protein, and of aldolase, phosphofructokinase, and lactate dehydrogenase proteins but not mRNA. This suggests that the modulation of glucose transport and glycolysis by hypoxia may contribute to the development of hypoxia-induced tolerance. In contrast, preconditioning with CoCl2 did not produce any change in HIF-1 target gene expression suggesting that different molecular mechanisms may be involved in the induction of tolerance by hypoxia and CoCl2 in newborn brain.

Inflammatory Cell Infiltration after Endothelin-1-Induced Cerebral Ischemia: Histochemical and Myeloperoxidase Correlation with Temporal Changes in Brain Injury

Accumulation of neutrophils in brain after transient focal stroke remains controversial with some studies showing neutrophils to be deleterious, whereas others suggest neutrophils do not contribute to ischemic injury. Myeloperoxidase (MPO) has been used extensively as a marker for quantifying neutrophil accumulation, but is an indirect method and does not detect neutrophils alone. To elucidate the interaction of macrophages in the neutrophil inflammatory response, we conducted double-label immunofluorescence in brain sections at 0, 1, 2, 3, 7, and 15 days after ischemia. Each of these results was obtained from the same animal to determine correlations between neutrophil infiltration and ischemic damage. It was found that MPO activity increased up to 3 days after cerebral ischemia. Dual-staining revealed that macrophages engulf neutrophils in the brain and that this engulfment of neutrophils increased with time, with 50% of neutrophils in the brain engulfed at 3 days and approximately 85% at 15 days (N=5, P < 0.05). Interestingly, at 7 days the amount of dual-staining was decreased to 20% (N=5, P < 0.05). Neutrophil infiltration was positively correlated with ischemic damage in both the cortex and striatum (r2 = 0.86 and 0.80, respectively, P < 0.01). The results of this study indicate that the MPO from neutrophils phagocytized by macrophages may continue to contribute to the overall MPO activity, and that previous assessments that have utilized this marker to measure neutrophil accumulation may have miscalculated the number of neutrophils within the ischemic territory and hence their contribution to the evolution of the infarct at later time points. Thus any biphasic infiltration of neutrophils may have been masked by the accumulation of macrophages.

Hypoxia-induced ischemic tolerance in neonatal rat brain involves enhanced ERK1/2 signaling.

Hypoxic preconditioning (HP) 24 h before hypoxic‐ischemic (HI) injury confers significant neuroprotection in neonatal rat brain. Recent studies have shown that the mitogen‐activated protein kinase (MAPK) and phosphatidylinositol‐3‐kinase (PI3K) intracellular signaling pathways play a role in the induction of tolerance to ischemic injury in heart and brain. To study the role of MAPK (ERK1/2, JNK, p38MAPK) and PI3K/Akt/GSK3β signaling pathways in hypoxia‐induced ischemic tolerance, we examined the brains of newborn rats at different time points after exposure to sublethal hypoxia (8% O2 for 3 h). Immunoblot analysis showed that HP had no effect on the levels of phosphorylated Akt, GSK3β, JNK and p38MAPK. In contrast, significantly increased levels of phosphorylated ERK1/2 were observed 0.5 h after HP. Double immunofluorescence staining showed that hypoxia‐induced ERK1/2 phosphorylation was found mainly in microvessels throughout the brain and in astrocytes in white matter tracts. Inhibition of hypoxia‐induced ERK1/2 pathway with intracerebral administration of U0126 significantly attenuated the neuroprotection afforded by HP against HI injury. These findings suggest that activation of ERK1/2 signaling may contribute to hypoxia‐induced tolerance in neonatal rat brain in part by preserving vascular and white matter integrity after HI.

Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: Evidence for delayed up-regulation and redistribution of GLT-1.

Excitatory amino acid transporters (EAATs) are responsible for homeostasis of extracellular L-glutamate, and the glial transporters are functionally dominant. EAAT expression or function is altered in acute and chronic neurological conditions, but little is known about the regulation of EAATs in reactive astroglia found in such neuropathologies. These studies examined the effects of the bacterial endotoxin lipopolysaccharide (LPS) on glial EAATs in vitro. The effects of LPS (1 microg/ml, 24-72 h) on EAAT activity and expression were examined in primary cultures of mouse astrocytes. [(3)H]D-aspartate uptake increased to 129% of control by 72 h treatment with LPS. Saturation analysis revealed that apparent K(m) was unchanged whilst V(max) was significantly increased to 172% of control by 72 h LPS treatment. Biotinylation and Western blotting indicated that cell-surface expression of GLT-1 was significantly elevated (146% control) by LPS treatment whereas GLAST expression was unchanged. Confocal analyses revealed that LPS treatment resulted in cytoskeletal changes and stellation of astrocytes, with rearrangement of F-actin (as shown by phalloidin labelling). Immunocytochemistry revealed clustering of GLAST, and increased expression and redistribution of GLT-1 to the cell-surface following treatment with LPS. Similar experiments were conducted in microglia, where LPS (50 ng/ml) was found to up-regulate expression of GLT-1 at 24 and 72 h in concert with cytoskeletal changes accompanying activation. These findings suggest an association of cytoskeletal changes in glia with EAAT activity, with the predominant adaptation involving up-regulation and redistribution of GLT-1.

Direct visualization of cholecystokinin subtype2 receptors in rat central nervous system using anti-peptide antibodies.

The cholecystokinin receptor, subtype 2 (CCK(2)R), is considered, based on receptor autoradiography, to be the predominant receptor for this peptide transmitter in the mammalian central nervous system. To directly visualize the CCK(2)R we utilized a convenient and sensitive immunohistochemical procedure using antipeptide receptor antibodies raised in rabbits against unique portions of the carboxyl tail and third intracellular loop of the CCK(2)R. Antibodies were characterized by ELISA and Western blotting, and used for immunohistochemistry in rat brain sections. Studies with both antibodies revealed a widespread topographic distribution of CCK(2)R-like immunoreactivity (CCK(2)R-LI) in regions such as cortex, olfactory bulb, nucleus accumbens, septum, striatum, hippocampus, basolateral amygdala, habenula, hypothalamus, thalamus, ventral mesencephalon, inferior colliculus, parabrachial nucleus, pontine nucleus, supercolliculus, red nucleus, subcommisural and occulomotor nucleus, area postrema, solitary, olivary, cochlear, cuneate and trigeminal nuclei and spinal cord dorsal horn in agreement with the results of previous receptor autoradiography.

Hypoxic preconditioning in neonatal rat brain involves regulation of excitatory amino acid transporter 2 and estrogen receptor alpha

Exposure of the brain to a sublethal insult can protect against a subsequent brain injury. Hypoxic preconditioning induces tolerance to hypoxic--ischemic injury in neonatal rat brain and is associated with changes in gene and protein expression. To study the involvement of excitatory amino acid transporters (EAAT1 and EAAT2) and estrogen receptors (ERalpha and ERbeta) in neonatal hypoxia--induced ischemic tolerance, we examined changes in expression of these proteins in the cortex, hippocampus and striatum of newborn rats at different time points after exposure to sublethal hypoxia (8% O(2), 3h). Preconditioning with hypoxia 24h before hypoxia-ischemia afforded marked brain protection compared with littermate control animals as determined by morphological assessment. Immunoblot analysis showed that EAAT2 and ERalpha were significantly increased by 55% and 49%, respectively, in cortex at 24h after hypoxic-preconditioning. Surprisingly, at the same time point, a significant decrease of EAAT2 by 48% in striatum was observed. In contrast, hypoxic preconditioning had no effect on the levels of EAAT1 and ERbeta in any of the brain regions studied at any of the time points analyzed. The similar pattern of changes in EAAT2 and ERalpha levels suggests that ERalpha might interact with EAAT2 in producing preconditioning. The endogenous molecular mechanisms modulated by hypoxia preconditioning may contribute to the development of hypoxia-induced ischemic tolerance, and may provide novel therapeutic targets for the treatment of cerebral ischemia.

Roles for Nitric Oxide as an Intra‐ and Interneuronal Messenger at NMDA Release‐Regulating Receptors: Evidence from Studies of the NMDA‐Evoked Release of [3H]Noradrenaline and d‐[3H]Aspartate from Rat Hippocampal Slices

Abstract: N‐Methyl‐d‐aspartate (NMDA) receptors regulating the release of [3H]noradrenaline ([3H]NA) and d‐[3H]aspartate (d‐[3H]Asp) were investigated in superfused slices of rat hippocampus in the presence and absence of nitrergic drugs to examine a possible role for nitric oxide (NO) in the release process. In Mg2+‐free Krebs‐Henseleit buffer, the NMDA‐evoked release of [3H]NA and d‐[3H]Asp was Ca2+ dependent and inhibited by the NMDA antagonist (±)‐3‐(2‐carboxypiperazin‐4‐yl)propenyl‐1‐phosphonic acid. NMDA‐stimulated release of [3H]NA was tetrodotoxin (TTX; 0.1–2 µM) sensitive, whereas that for d‐[3H]Asp was TTX insensitive, indicating that the NMDA receptors involved are differentially localized; those for d‐[3H]Asp appear to be presynaptic, whereas those for [3H]NA are extrasynaptic in location. l‐Arginine (100 µM), the natural precursor of NO synthesis, enhanced NMDA‐evoked release of [3H]NA (100%) and d‐[3H]Asp (700%). Exogenous NO donors—sodium nitroprusside, 3‐morpholinosyndnomine, and S‐nitroso‐N‐acetylpenicillamine (all 100 µM)—stimulated the NMDA‐evoked release. An exception was the inhibition by nitroprusside of NMDA‐evoked release of [3H]NA, where the presence of antioxidants may influence channel activity. Inhibitors of NO synthase (NG‐nitro‐, NG‐methyl‐, and NG‐amino‐l‐arginine, all 100 µM) attenuated (50–80%) the NMDA‐stimulated release of [3H]NA and d‐[3H]Asp, as did KN‐62 (10 µM), a specific inhibitor of calmodulin kinase II. Our data support roles for the NO transducing system subsequent to the activation of NMDA release‐regulating receptors as both an intraneuronal (presynaptically) and an extraneuronal messenger.

Long-Term Functional and Protective Actions of Preconditioning With Hypoxia, Cobalt Chloride, and Desferrioxamine Against Hypoxic-Ischemic Injury in Neonatal Rats

Preconditioning with hypoxia and hypoxia-mimetic compounds cobalt chloride (CoCl2) and desferrioxamine (DFX) protects against hypoxic-ischemic (HI) injury in neonatal rat brain. We examined long-term functional and protective actions of preconditioning induced by hypoxia, CoCl2 and DFX in a neonatal rat model of HI. Postnatal day six rat pups were exposed to preconditioning with hypoxia (8% oxygen) or injections of CoCl2, DFX or saline vehicle and 24 h later rats underwent HI or sham surgery. Behavioral tests were performed and at the conclusion of experiments, brains removed for morphologic analyses. HI resulted in a large unilateral lesion in the ipsilateral hemisphere compared with sham control rats. All preconditioning treatments significantly reduced the total lesion volume. Behavioral deficits were observed in HI rats compared with sham controls. The reduction in forelimb grasping strength in HI rats was attenuated by preconditioning with hypoxia, CoCl2 and DFX. HI increased the number of foot faults in a grid-walking test and resulted in forelimb asymmetry in the cylinder test. Only preconditioning with hypoxia reversed all three functional deficits after HI. These findings indicate that preconditioning, especially when induced by hypoxia, has the potential to minimize the morphologic and functional effects of neonatal HI injury.

Hypoxic preconditioning produces differential expression of hypoxia-inducible factor-1α (HIF-1α) and its regulatory enzyme HIF prolyl hydroxylase 2 in neonatal rat brain

Hypoxic preconditioning can protect the brain against a subsequent damaging ischaemic insult. Mild hypoxia alone seems not sufficient to cause neuronal injury, but can induce changes in gene expression and intracellular signalling pathways and the hypoxia-inducible transcription factor (HIF-1) is a key modulator of these genes. Recently, a family of HIF prolyl hydroxylase enzymes (PHDs) has been shown to regulate HIF-1 function by controlling its degradation. Since PHD-2 is thought to be the predominant isoform which regulates HIF-1, we have investigated whether preconditioning with hypoxia can affect levels of PHD-2 and HIF-1alpha proteins to elucidate roles for the HIF-1/PHD-2 system in the neuroprotection conferred by hypoxic preconditioning. Sprague-Dawley rats (postnatal Day 6 (p6)) were exposed to preconditioning with hypoxia (3 h, 8% oxygen) or normoxia (3 h, room air) at various times (0, 0.5, 2, 4, 16 and 24 h) after reoxygenation, brains were obtained for Western blot and immunohistochemical analyses of PHD-2 and HIF-1alpha proteins. Western blotting studies demonstrate a significant increase in the expression of PHD-2 ( approximately 1.8-2-fold increase, at 0.5, 16 and 24 h after reoxygenation; p < 0.01) and HIF-1alpha (approximately 1.7-fold increase immediately after hypoxia; p < 0.05) proteins following hypoxic preconditioning relative to normoxic control tissue. Similar results were observed in immunohistochemical studies examining PHD-2 and HIF-1alpha proteins. Our study demonstrated for the first time that in vivo exposure to systemic hypoxia elevates the expression of PHD-2 protein in brain and it is likely that enhancing HIF-1 function by inhibition of PHD activity is involved in the protective effect conferred by hypoxic preconditioning in neonatal rat brain.

Injury to axons and oligodendrocytes following endothelin-1-induced middle cerebral artery occlusion in conscious rats

Injury to axons and oligodendrocytes has been poorly characterized in most animal models of stroke, and hence has been difficult to target therapeutically. It is therefore necessary to characterize axonal and oligodendroglial injury in these models, in order to rationally design putative protective compounds that minimize this injury. This study aims to characterize injury to axons and oligodendrocytes in the endothelin-1 (ET-1) model of middle cerebral artery occlusion (MCAO) in conscious rats. Transient forebrain ischemia was induced in conscious adult male Long Evans rats by the perivascular microinjection of ET-1. Quantitative histopathology was performed on forebrain sections at 6, 24, 48 and 72 h after ET-1 administration, using ballistic light analyses and immunohistochemistry for amyloid precursor protein (APP), SMI32, and Tau-1. Ballistic light analyses of cortical and striatal lesions revealed that the infarct volume was maximal in these regions by 6 h. APP and SMI32 immunohistochemistry demonstrated that axonal injury was maximal by 6 h in this model; however, some injured axons appeared to maintain good structural integrity up to 72 h after insult. Density measurements for Tau-1-immunopositive oligodendrocytes were significantly elevated within the corpus callosum from 48 h, but reductions in total oligodendrocyte numbers were not apparent up 72 h after ET-1 injection. These results indicate that axonal and oligodendroglial injury should be investigated as potential targets for delayed therapeutic intervention after MCAO.