M Urbanek

Erythropoietin is neuroprotective against NMDA-receptor-mediated excitotoxic brain injury in newborn mice

Using an established mouse model of human periventricular leukomalacia, we investigated whether EPO could reduce excitotoxic damage. When administered 1 h following intracerebral injection of 10 microg ibotenic acid at day 5 of life, both a single injection of EPO (5000 IU/kg bw) and repetitive administrations of EPO reduced white and gray matter lesion size. The therapeutic window for protection was small as the protective effect of EPO was lost when EPO administration was delayed to 4 h post-insult. EPO-mediated upregulation of EPO-R, but not EPO, mRNA was observed within 4 h of the excitotoxic insult. The EPO effect was gender independent. Minor hematopoetic effects were observed following EPO treatment. We conclude that a single dose of EPO is sufficient to reduce excitotoxic brain injury and may therefore possess therapeutic relevance in the clinical setting.

Neuroprotective effects of the sigma-1 receptor ligand PRE-084 against excitotoxic perinatal brain injury in newborn mice.

Excessive glutamate release followed by N-methyl-D-aspartate receptor (NMDAR) activation plays a crucial role in perinatal brain injury. We have previously shown that dextromethorphan, a low-affinity NMDAR antagonist with anti-inflammatory properties, is neuroprotective against neonatal excitotoxic brain injury. Of interest, dextromethorphan is also a sigma-1 receptor (σ1R) agonist. The pharmacologic class of σ1R agonists has yielded propitious results in various animal models of adult central nervous system pathology. In an established neonatal mouse model of excitotoxic brain injury, we evaluated the effect of the selective σ1R agonist 2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate (PRE-084). A single intraperitoneal injection of 0.1 μg/g (low dose) or 10 μg/g (high dose) bodyweight (bw) PRE-084, given 1h after the excitotoxic insult, significantly reduced lesion size in cortical gray matter 24 h and 120 h after the insult. Repetitive injections of 0.1 μg/g PRE-084 proved to be equally effective. PRE-084 treatment resulted in a decrease in cell death indicated by reduced TUNEL positivity and caspase-3 activation. Furthermore, it lowered the number of isolectin B4-positive, activated microglial cells. PRE-084 had no effect on developmental apoptosis in the undamaged brain. In vitro findings in primary hippocampal neurons suggest that PRE-084 treatment provides partial protection against glutamate induced morphological and functional changes. For excitotoxicity as playing a crucial role in the pathogenesis of perinatal brain injury, we demonstrate for the first time that systemic treatment with the highly selective σ1R agonist PRE-084 protects against NMDAR-mediated excitotoxic brain damage.

Dextromethorphan is protective against sensitized N‐methyl‐d‐aspartate receptor‐mediated excitotoxic brain damage in the developing mouse brain

Enhanced glutamate release and inflammation play an important role in the pathogenesis of developmental brain injury. Although N‐methyl‐d‐aspartate receptor (NMDAR) antagonists potently attenuate neonatal brain damage in several animal models, they can also impact trophic functions in the developing brain. As a consequence, high‐affinity NMDAR antagonists have been shown to trigger widespread apoptotic neurodegeneration in the newborn brain. Dextromethorphan (DM), a low‐affinity NMDAR antagonist with anti‐inflammatory properties, may be neuroprotective against excitotoxic and inflammation‐enhanced excitotoxic brain injury, without the associated stimulation of apoptotic degeneration. Using an established newborn mouse model of excitotoxic brain damage, we determined whether systemic injection of DM significantly attenuates excitotoxic lesion size. We investigated several doses and time regimens; a dose of 5 µg/g DM given in a combination of both pre‐injury and repetitive post‐injury treatment proved most effective. DM treatment significantly reduced lesion size in gray and white matter by reducing cell death as shown by a decreased Fluoro‐Jade B staining and caspase‐3 activation. Pre‐treatment with interleukin‐1β and lipopolysaccharide enhanced NMDAR‐mediated excitotoxic brain injury and microglial cell activation. This sensitizing effect was abolished by DM treatment, as the effectiveness of DM in reducing lesion size and microglial cell activation was similar to phosphate‐buffered saline‐pre‐treated controls. In all cases, no gender‐specific differences were detected. DM treatment did not trigger any apoptotic neurodegeneration (caspase‐3 cleavage, terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling, Fluoro‐Jade B staining). Although functional parameters were not measured, our data corroborate reports that DM is neuroprotective and that it may therefore improve functional outcome following perinatal brain injury.

Systemic application of granulocyte-colony stimulating factor and stem cell factor exacerbates excitotoxic brain injury in newborn mice

Granulocyte-colony stimulating factor (G-CSF) has been shown to reduce brain lesion size and mortality in adult mice after hypoxic-ischemic injury. Another hematopoietic growth factor, stem cell factor (SCF), has been shown to be up-regulated in the brains of adult rodents following brain damage, where it stimulates postlesional neurogenesis. Injection of the excitotoxic agent ibotenate into the brain of newborn mice produces a brain lesion characterized by neuronal death and white matter cysts, which is similar to periventricular leucomalacia. The aim of the present study was to investigate whether administration of SCF and G-CSF is neuroprotective against ibotenate lesions in neonatal mice. Contrary to our expectations, cortical and white matter brain lesions induced by ibotenate were enhanced following the administration of 50 μg/kg SCF or 200 μg/kg G-CSF. Dose-response studies indicated that G-CSF could increase grey matter lesions even at lower dosages (22 and 66 μg/kg). Administration of SCF and G-CSF in combination also increased cortical and white matter lesions, to 133 ± 8% and 187 ± 12%. In the undamaged brain, G-CSF or G-CSF+SCF treatment had no effect on apoptosis in the grey or white matter; however, these treatments significantly increased apoptosis in the damaged brain. Our data clearly demonstrate that G-CSF and SCF are not neuroprotective and result in deleterious enhancement of excitotoxic brain damage in newborn mice. We conclude that G-CSF and SCF should be used cautiously in newborn infants with brain lesions; if they are used, long term neurologic and neurodevelopmental follow-up is warranted.

Levetiracetam increases neonatal hypoxic-ischemic brain injury under normothermic, but not hypothermic conditions.

BACKGROUND Hypoxic-ischemic encephalopathy (HIE) resulting from perinatal asphyxia often leads to severe neurologic impairment or even death. There is a need to advance therapy for infants with HIE, for example to combine hypothermia with pharmacological treatment strategies. Levetiracetam (LEV) is approved for clinical administration to infants older than 4 weeks of age and is also used off-label in neonates. Furthermore, LEV was shown to be neuroprotective in adult animal models of brain injury. AIM OF THE STUDY The aim of this study was to evaluate the neuroprotective potential of LEV in vitro using primary hippocampal neurons, and in vivo using an established model of neonatal hypoxic-ischemic brain injury. RESULTS LEV treatment per se did not induce neurotoxicity in the developing rodent brain. Following oxygen glucose deprivation, we observed some, although not a significant, increase in cell death after LEV treatment. In vivo, LEV was administered under normothermic and hypothermic conditions following hypoxic-ischemic brain damage. LEV administration significantly increased brain injury under normothermic conditions. Compared to the normothermia-treated group, in the hypothermia group LEV administration did not increase hypoxic-ischemic brain injury. DISCUSSION This study demonstrates that LEV treatment increases neonatal hypoxic-ischemic brain injury. Administration of LEV in the acute phase of the injury might interfere with the balanced activation and inactivation of excitatory and inhibitory receptors in the developing brain. The neurotoxic effect of LEV in the injured newborn brain might further suggest an agonistic effect of LEV on the GABAergic system. Hypothermia treatment attenuates glutamate release following hypoxic-ischemic brain injury and might therefore limit the potentially deleterious effects of LEV. As a consequence, our findings do not necessarily rule out a potentially beneficial effect, but argue for cautious use of LEV in newborn infants with pre-existing brain injury.

The sigma-1 receptor agonist 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) protects against newborn excitotoxic brain injury by stabilizing the mitochondrial membrane potential in vitro and inhibiting microglial activation in vivo

Premature birth represents a clinical situation of risk for brain injury. The diversity of pathophysiological processes complicates efforts to find effective therapeutic strategies. Excitotoxicity is one important factor in the pathogenesis of preterm brain injury. The observation that sigma-1 receptor agonists possess neuroprotective potential, at least partly mediated by a variety of anti-excitotoxic mechanisms, has generated great interest in targeting those receptors to counteract brain injury. The objective of this study was to evaluate the effect of the highly specific sigma-1 receptor agonist, 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) to protect against excitotoxic developmental brain injury in vivo and in vitro. Primary hippocampal neurons were pre-treated with PPBP before glutamate was applied and subsequently analyzed for cell death (PI/calcein AM), mitochondrial activity (TMRM) and morphology of the neuronal network (WGA) using confocal microscopy. Using an established neonatal mouse model we also determined whether systemic injection of PPBP significantly attenuates excitotoxic brain injury. PPBP significantly reduced neuronal cell death in primary hippocampal neurons exposed to glutamate. Neurons treated with PPBP showed a less pronounced loss of mitochondrial membrane potential and fewer morphological changes after glutamate exposure. A single intraperitoneal injection of PPBP given one hour after the excitotoxic insult significantly reduced microglial cell activation and lesion size in cortical gray and white matter. The present study provides strong support for the consideration of sigma-1 receptor agonists as a candidate therapy for the reduction of neonatal excitotoxic brain lesions and might offer a novel target to counteract developmental brain injury.

Role of p75NTR in NMDAR-mediated excitotoxic brain injury in neonatal mice.

BACKGROUND Perinatal brain injury in preterm infants is a major cause of neurological handicap. The role of the neurotrophin receptor p75 (p75(NTR)) in the pathogenesis and repair of neonatal excitotoxic brain injury is unknown. Depending on a complex interplay of neurotrophin signalling, p75(NTR) can, in addition to its trophic function, also induce apoptosis. HYPOTHESIS We hypothesised that excitotoxicity increases p75(NTR) expression and p75(NTR) knockout (KO) mice have a significantly smaller lesion size upon excitotoxicity as compared to wild-type (WT) mice. METHODS We used an established animal model of neonatal excitotoxic brain injury mimicking several key aspects of human preterm brain damage. We subjected five-day-old WT and KO mice to excitotoxic injury by means of a single intracranial ibotenate injection (N-methyl-D-aspartate receptor agonist, NMDAR) into one brain hemisphere. Lesion size, number of activated caspase-3- and apoptosis-inducing factor (AIF)-positive cells were determined as outcome parameters. Gender analyses were taken into account retrospectively. RESULTS NMDAR-mediated excitotoxicity induced an upregulation of p75(NTR) expression in the peri-lesion area. Lesion size was significantly increased in female KO as compared to male KO animals. Knockout of p75(NTR) reduced the number of activated caspase-3 but not AIF-positive cells after NMDAR-mediated excitotoxic injury independently of gender. CONCLUSION Since NMDAR-mediated excitotoxic brain injury induced p75(NTR) expression and caspase-3-activated apoptosis in p75(NTR) KO animals was decreased, we conclude that activation of p75(NTR) contributes to NMDAR-mediated apoptosis in the neonatal brain. An increase in lesion size in female animals after excitotoxic brain injury suggests that in females p75(NTR) seems to play a dual role.

The common antitussive agent dextromethorphan protects against hyperoxia-induced cell death in established in vivo and in vitro models of neonatal brain injury

Preterm infants are prematurely subjected to relatively high oxygen concentrations, even when supplemental oxygen is not administered. There is increasing evidence to show that an excess of oxygen is toxic to the developing brain. Dextromethorphan (DM), a frequently used antitussive agent with pleiotropic mechanisms of action, has been shown to be neuroprotective in various models of central nervous system pathology. Due to its numerous beneficial properties, it might also be able to counteract detrimental effects of a neonatal oxygen insult. The aim of the current study was to evaluate its therapeutic potential in established cell culture and rodent models of hyperoxia-induced neonatal brain injury. For in vitro studies pre- and immature oligodendroglial (OLN-93) cells were subjected to hyperoxic conditions for 48 h after pre-treatment with increasing doses of DM. For in vivo studies 6-day-old Wistar rat pups received a single intraperitoneal injection of DM in two different dosages prior to being exposed to hyperoxia for 24h. Cell viability and caspase-3 activation were assessed as outcome parameters at the end of exposure. DM significantly increased cell viability in immature oligodendroglial cells subjected to hyperoxia. In pre-oligodendroglial cells cell viability was not significantly affected by DM treatment. In vivo caspase-3 activation induced by hyperoxic exposure was significantly lower after administration of DM in gray and white matter areas. In control animals kept under normoxic conditions DM did not significantly influence caspase-3-dependent apoptosis. The present results indicate that DM is a promising and safe treatment strategy for neonatal hyperoxia-induced brain injury that merits further investigation.

Administration of secretoneurin is protective in hypoxic–ischemic neonatal brain injury predominantly in the hypoxic-only hemisphere

Neonatal brain injury is a problem of global importance. To date, no causal therapies are available. A substance with considerable therapeutic potential is the endogenous neuropeptide secretoneurin (SN), which has proven to be beneficial in adult stroke. The aim of this study was to assess its effect in neonatal hypoxic-ischemic brain injury models. In vitro, primary hippocampal neurons were pre-treated with vehicle, 1µg/ml, 10µg/ml, or 50µg/ml SN and subjected to oxygen-glucose deprivation (OGD) for six hours. Cell death was assessed after a 24-h recovery period. In vivo, seven day-old CD-1 mice underwent unilateral common carotid artery ligation and were exposed to 8% oxygen/nitrogen for 20 min. SN plasma concentrations were serially determined by ELISA after insult. One hour after hypoxia, a subgroup of animals was treated with vehicle or SN. SN plasma concentrations significantly decreased 48h after insult. The number of caspase-3-positive cells was significantly lower in the hypoxic-ischemic hemisphere in the thalamus of SN-treated animals. In the hypoxic-only hemisphere administration of SN significantly reduced the number of caspase-3-positive cells (in cortex, white matter, hippocampus, thalamus and striatum) and inhibited microglial cell activation in the thalamus. SN has neuroprotective potential in neonatal brain injury. Its main action seems to be inhibition of apoptosis in the aftermath of the insult, predominantly in the hypoxic-only hemisphere. This might be explained by the less pronounced injury in this hemisphere, where blood flow and thus nutrient supply are maintained.

Delayed application of the haematopoietic growth factors G-CSF/SCF and FL reduces neonatal excitotoxic brain injury

BACKGROUND Developmental brain injury results in cognitive and motor deficits in the preterm infant. Enhanced glutamate release and subsequent receptor activation are major pathogenetic factors. The effect of haematopoietic growth factors, such as granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF) and flt-3 ligand (FL) on neonatal brain injury is controversially discussed. Timing of treatment is known to be a crucial factor. Based on the hypothesis that an exacerbation of injury is caused by administration of substances in the acute phase, the objective of this study was to evaluate the effect of delayed administration of G-CSF/SCF and FL to protect against excitotoxic brain injury in vivo. METHODS In an established neonatal mouse model of excitotoxic brain injury, we evaluated the effect of daily intraperitoneal doses of G-CSF/SCF or FL, starting 60 h after the excitotoxic insult. RESULTS Intraperitoneal injections of G-CSF/SCF and FL, given 60 h after the excitotoxic insult, significantly reduced lesion size at postnatal days 10, 18 and 90. G-CSF/SCF treatment resulted in a decrease in apoptotic cell death indicated by reduced caspase-3 activation. G-CSF/SCF and FL treatment did not affect apoptosis-inducing factor-dependent apoptosis or cell proliferation. CONCLUSION We show that delayed systemic treatment with the haematopoietic growth factors G-CSF/SCF and FL protects against N-methyl-D-aspartate receptor-mediated developmental excitotoxic brain damage. Our results suggest that neuroprotective effects in this neonatal animal model of excitotoxic brain injury depend on the timing of drug administration after the insult.