Cerebral Palsy

Copper deficiency is associated with impaired brain development and mitochondrial dysfunction. Perinatal copper deficiency was produced in Holtzman rats. In vivo proton NMR spectroscopy was used to quantify 18 cerebellar and hippocampal metabolites on postnatal day 21 (P21). Copper status was evaluated in male copper-adequate (CuA) and copper-deficient (CuD) brothers at P19 and at P23, 2 days following NMR experiments, by metal and in vitro metabolite data. Compared to CuA pups, CuD pups had lower ascorbate concentration in both brain regions, confirming prior HPLC data. Both regions of CuD rats also had lower N-acetylaspartate levels consistent with delayed development or impaired mitochondrial function similar to prior work demonstrating elevated lactate and citrate. For other metabolites, the P21 neurochemical profile of CuD rats was remarkably similar to CuA rats but uniquely different from iron-deficient or chronic hypoxia models. Further research is needed to determine the neurochemical consequences of copper deficiency.

Hypoxia-ischemia constitutes a risk in infants by altering cerebral blood flow regulatory mechanisms and causing loss of cerebral vascular auto-regulation. Hypotension, cerebral ischemia, and reperfusion are the main events involved in vascular auto-regulation leading to cell death and tissue damage. Reperfusion could be critical since organ damage, particularly of the brain, may be amplified during this period. An exaggerated activation of vasoactive agents of calcium mediated effects could be responsible for reperfusion injury, which, in turns, leads to cerebral hemorrhage and damage. These dramatic phenomena represent a common repertoire in infants complicated by perinatal acute or chronic hypoxia or cardiovascular disorders treated by risky procedures such as open heart surgery and cardiopulmonary by-pass (CPB). To date, despite accurate perinatal and intra-operative monitoring, the post-insult period is crucial, since clinical symptoms and monitoring parameters may be of no avail and therapeutic window for pharmacological intervention (6-12 hours) may be limited, at a time when brain damage is already occurring. Therefore, the measurement of circulating biochemical markers of brain damage, such as vasoactive agents and nervous tissue peptides is eagerly awaited in clinical practice to detect high risk infants. The present review is aimed at investigating the role as circulating biochemical markers such as adrenomedullin, a vasoactive peptide; S100B, a calcium binding protein, activin A, a glycoprotein; neuronal specific enolase (NSE), a dimeric isoenzyme; glial fibrillary acid protein (GFAP), a astroglial protein, in the cascade of events leading to ischemia reperfusion injury in infants complicated by perinatal asphyxia or cardiovascular disorders requiring risky therapeutic strategies such as CPB and/or extracorporeal membrane oxygenation.

The existence of a primitive, elemental form of CNS arousal, “generalized arousal,” has been hypothesized; it has been given an operational definition, and a high throughput assay has been assayed for it in mice. Many of the ascending and descending neuroanatomical pathways are fairly well understood. To begin experiments that might have potential implications for therapeutic measures, mice were rendered anoxic and it was shown that the assay can demonstrate behavioral abnormalities not detected with a standard neurological screen. These behavioral deficiencies are reminiscent of “sundowning,” a form of dementia seen in hospitalized elderly patients. Electrical stimulation of neurons in medial thalamic cell groups can increase activity in the generalized arousal assay. Current studies include attempts to achieve temporal patterns of electrical stimulation that would take advantage of nonlinear properties of ascending arousal pathways.

Preterm birth results in significant neurodevelopmental disability. A neonatal rodent model of chronic sublethal hypoxia (CSH), which mimics effects of preterm birth, was used to characterize neurodevelopmental consequences of prolonged exposure to hypoxia using tissue anisotropy measurements from diffusion tensor imaging. Corpus callosum, cingulum, and fimbria of the hippocampus revealed subtle, yet significant, hypoxia-induced modifications during maturation (P15-P51). Anisotropy differences between control and CSH mice were greatest at older ages (>P40) in these regions. Neither somatosensory cortex nor caudate putamen revealed significant differences between control and CSH mice at any age. We assessed control and CSH mice using tests of general activity and cognition for behavioral correlates of morphological changes. Open-field task revealed greater locomotor activity in CSH mice early in maturation (P16-P18), whereas by adolescence (P40-P45) differences between control and CSH mice were insignificant. These results may be associated with lack of cortical and subcortical anisotropy differences between control and CSH mice. Spatial-delayed alternation and free-swim tasks in adulthood revealed lasting impairments for CSH mice in spatial memory and behavioral laterality. These differences may correlate with anisotropy decreases in hippocampal and callosal connectivities of CSH mice. Thus, CSH mice revealed developmental and behavioral deficits that are similar to those observed in low birth weight preterm infants.

Hypoxia promotes Na,K-ATPase endocytosis via PKCzeta-mediated phosphorylation of its alpha subunit. Here, we describe that hypoxia leads to phosphorylation of AMPK at Thr172 in rat alveolar epithelial cells. Over-expression of a dominant-negative AMPKalpha construct prevented the hypoxia-induced endocytosis of Na,K-ATPase. Overexpression of the reactive oxygen species (ROS) scavenger catalase prevented the hypoxia-induced AMPK activation. Moreover, hypoxia failed to activate AMPK in mitochondria-deficient A549-rho(0) cells, suggesting that mitochondrial ROS play an essential role in the hypoxia-induced AMPK activation. The hypoxia-induced PKCzeta translocation to the plasma membrane and phosphorylation at Thr410 was prevented by pharmacologically inhibiting AMPK or by over-expression of AMPK-DN construct. We found that AMPKalpha phosphorylates PKCzeta on residue Thr410 within the PKCzeta activation loop. Importantly, activation of AMPKalpha was necessary for hypoxia-induced AMPK-PKCzeta binding in alveolar epithelial cells. Overexpression of mutant PKCzeta-T410A prevented the hypoxia-induced Na,K-ATPase endocytosis, confirming that PKCzeta Thr410 phosphorylation is essential for this process. PKCzeta activation by AMPK is isoform specific as siRNA against the alpha1 but not the alpha2 catalytic subunit prevented PKCzeta activation. Accordingly, we provide first evidence that hypoxia-generated mitochondrial ROS lead to the activation of the AMPK alpha1 isoform, which binds and directly phosphorylates PKCzeta at Thr410, thereby promoting Na,K-ATPase endocytosis.

Neuroglobin is a recently identified globin molecule that is expressed predominantly in the vertebrate brain. Neuroglobin expression increases in oxygen-deprived neurons, suggesting it protects neurons from ischemic cell death. We report that neuroglobin transcript and protein are expressed in human glioblastoma cells, and that this expression increases in hypoxia in vitro. We also show that neuroglobin is up-regulated in hypoxic microregions of glioblastoma tumor xenografts. Our finding of hypoxic up-regulation of neuroglobin in human glioblastoma cells may provide insight into how tumor cells adapt to and survive in hypoxic microenvironments.

OBJECTIVE:: In this study, available medical literature were reviewed to determine whether brain hypoxia as measured by brain tissue oxygen (Bto2) levels is associated with increased risk of poor outcome after traumatic brain injury (TBI). A secondary objective was to examine the safety profile of a direct BtO2 probe. DATA SOURCE AND EXTRACTION:: Clinical studies published between 1993 and 2008 were identified from electronic databases, Index Medicus, bibliographies of pertinent articles, and expert consultation. The following inclusion criteria were applied for outcome analysis: 1) more than 10 patients described, 2) use of a direct Bto2 monitor, 3) brain hypoxia defined as Bto2 <10 mm Hg for >15 or 30 minutes, 4) 6-month outcome data, and 5) clear reporting of patient outcome associated with Bto2. For the analysis, each selected article had to have adequate data to determine odds ratios (ORs) and confidence intervals (CIs). Thirteen studies met the initial inclusion criteria and three were included in the final outcome analysis. Safety data were abstracted from any report where it was mentioned. DATA SYNTHESIS:: The three studies included 150 evaluable patients with severe TBI (Glasgow Coma Scale </=8). Brain hypoxia was identified in 71 (47%) of these patients. Among the patients with brain hypoxia, 52 (73%) had unfavorable outcome including 39 (55%) who died. In the absence of brain hypoxia, 34 (43%) patients had an unfavorable outcome, including 17 (22%) who died. Overall brain hypoxia (Bto2 <10 mm Hg >15 minutes) was associated with worse outcome (OR 4.0; 95% CI 1.9-8.2) and increased mortality (OR 4.6; 95% CI 2.2-9.6). We reviewed published safety data; in 292 patients monitored with a Bto2 probe, only two adverse events were reported. CONCLUSION:: Summary results indicate that brain hypoxia (<10 mm Hg) is associated with worse outcome after severe TBI and that Bto2 probes are safe. These results imply that treating patients to increase Bto2 may improve outcome after severe TBI. This question will require further study.

Ischemic brain injury is a dynamic process that involves oxidative stress, inflammation, and cell death, as well as activation of endogenous adaptive and regenerative mechanisms depending on activation of transcription factors such as hypoxia inducible factor 1-alpha (HIF-1alpha). Because CoCl(2) activates HIF-1alpha, we described a new focal-hypoxia model by direct intracerebral CoCl(2) injection. Adult male Wistar rats were intracerebrally injected with CoCl(2) (2 mul-50 mM), in frontoparietal cortex of right hemisphere, and saline (2 mul) in the contralateral hemisphere. In slides of fixed brains at 1, 6, 9, 24 h or 5 day after treatment, TTC, histochemistry (toluidine blue, Hoescht-33342, TUNEL), immunostaining (HIF-1alpha, GFAP), Lycopersicon esculentum lectin staining, and electron microscopy (EM) were performed. Immediately after 1 h post CoCl(2) injection, HIF-1alpha stabilization and neuronal nuclear shrinkage and cromathin condensation were observed by immunostaining and EM, respectively. Neuronal apoptotic nuclear morphology and GFAP immunoreactivity and lectin maximal reactivity were detected during 6-9 h. Ultrastructural alterations of morphology included edematous perinuclear cytoplasm, organelles and endoplasmic reticulum (RE) enlargement, mitochondrial swelling with increased matrix density, and deposits of electron-dense material. Neurons showed particular nuclear indentations. Astrocytes and oligodendrocytes presented alterations in both nuclei and RE with dilated lumen and altered mitochondrias, and all these ultrastructural changes became detectable at day 5. CoCl(2) cortical injection mimics focal brain ischemia, inducing neuronal death and glial activation. This model brings the opportunity to develop focal ischemia in selected brain areas to study their functional consequences and potential pharmacological therapies for in vivo models of stroke.

Several protein phosphatases are involved in neuroprotection in response to ischemic brain injury. Here, we report that reactive oxygen species (ROS)-mediated oxidative stress promotes phosphorylation of endogenous SHP-2 through lipid rafts in rat primary astrocytes. SHP-2 was transiently phosphorylated during hypoxia/reoxygenation, an effect abrogated by a ROS scavenger and an NADPH oxidase inhibitor. Additionally, exogenous treatment with hydrogen peroxide (H(2)O(2)) triggered SHP-2 phosphorylation in a time- and dose-dependent manner and led to its translocation into lipid rafts. H(2)O(2)-mediated SHP-2 phosphorylation and translocation were inhibited by filipin III and methyl-beta-cyclodextrin (MCD), lipid-raft-disrupting agents. In the presence of H(2)O(2), SHP-2 formed a complex with STAT-3 and reduced the steady-state STAT-3 phosphorylation level. Interestingly, the effect of H(2)O(2) on SHP-2 phosphorylation was cell-type specific. Remarkably, SHP-2 phosphorylation was induced strongly by H(2)O(2) in astrocytes, but barely detectable in microglia. Our results collectively indicate that SHP-2 is activated by ROS-mediated oxidative stress in astrocytes and functions as a component of the raft-mediated signaling pathway that acts through dephosphorylation and inactivation of other phosphotyrosine proteins, such as STAT-3.

Neuroblasts produced by the neural stem cells of the adult subventricular zone (SVZ) migrate into damaged brain areas after stroke or other brain injuries, and previous data have suggested that they generate regionally appropriate new neurons. To classify the types of neurons produced subsequent to ischemic injury, we combined BrdU or virus labeling with multiple neuronal markers to characterize new cells at different times after the induction of stroke. We show that SVZ neuroblasts give rise almost exclusively to calretinin-expressing cells in the damaged striatum, resulting in the accumulation of these cells during long term recovery after stroke. The vast majority of SVZ neuroblasts as well as newly born young and mature neurons in the damaged striatum constitutively express the transcription factor Sp8, but do not express transcription factors characteristic of medium-sized spiny neurons, the primary striatal projection neurons lost after stroke. Our results suggest that adult neuroblasts do not alter their intrinsic differentiation potential after brain injury.