Karina Wegleiter

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.

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.

311 The Sigma-Receptor-Agonist 4-Phenyl-1-(4-Phenylbutyl) Piperidine (PPBP) Decreases Neonatal Excitotoxic Brain Injury and Diminishes Microglial Cell Activation

The pathogenesis of perinatal brain injury is multifactorial. In animal models of adult brain injury the sigma-1-receptor agonist 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) was shown to be neuroprotective due to modulation of inflammatory and apoptotic pathways and affection of excitotoxic signaling cascades. In the current study we evaluated the effect of PPBP in a neonatal animal model of excitotoxic brain injury. We performed an intracranial injection of ibotenate in CD1 mice on postnatal day 5 (P5) to induce excitotoxic injury. A single injection of vehicle or PPBP, in a dosage of 1, 5 or 50 µg/g body weight, was applied intraperitoneally one hour after the insult. Primary study endpoints were evaluated 24 (P6) and 120 (P10) hours after the insult by analysis of lesion size, number of cells stained positively for cleaved caspase-3 and isolectin B4. Systemic application of PPBP resulted in a significant reduction of lesion size in cortical gray matter in all dosages (p<0.05). and underlying white matter in 1 and 50µg/g (p<0.05) at P10. At P6 microglial cell activation was significantly diminished by PPBP in cortical gray (p<0,001) and underlying white matter (p<0.05). Lesion size on P6 and number of cells stained positively for activated caspase-3 at P6 was not affected by the treatment (p>0.05). In this neonatal animal model we were able to demonstrate a beneficial effect of the sigma-1 receptor agonist PPBP. This data suggest that PPBP may be a potential therapeutic approach in neonatal brain injury.

1651 Angiotensin II Receptor Antagonist Related Fetopathy - A Case Report

Fetal angiotensin II receptor antagonist exposure during pregnancy is associated with major congenital malformations including premature birth, oligohydramnios, acute renal failure, pulmonary hypoplasia and hypocalvaria. Fetopahty is mainly caused by renal insufficiency due to severe hypotension and disturbance of renal development. Therefore administration during the second and third trimester of pregnancy is contraindicated. We report on a 35 year-old woman with arterial hypertension who was referred to our obstetrical department because of oligohydramnios. She reported to receive treatment with Olmesartanmedoxomil (5mg/day). The condition resolved after changing anti-hypertensive treatment to metoprolol at 26 6/7 weeks of pregnancy. The patient was born at term by C-section and showed the following signs of fetopathy: hyperechogenic multicystic kidneys and hypocalvaria. Renal function was normal, nevertheless arterial hypertension was present but treatment was not required. Discharge from hospital was possible at the age of 9 days. Regularly follow-up visits are necessary to monitor renal function and to evaluate long term effects. Incidence of sartan-related fetopathy is unknown, therefore consistent reporting is mandatory. We present a case with mild presentation of symptoms, probably related to low therapeutic dosage and early change of antihypertensive treatment.

9 The Sigma-1 Receptor Agonist PRE-084 Attenuates Inflammation-Sensitized Nmdar-Mediated Excitotoxic Brain Injury in Newborn Mice

Excitotoxicity and inflammation play crucial roles in the etiopathogenesis of perinatal brain injury. We have shown that the sigma-1 receptor agonist 2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate (PRE-084) protects against N-methyl-d-aspartate (NMDA) receptor-mediated excitotoxic brain injury. In models of adult central nervous system pathology, PRE-084 has demonstrated potent anti-inflammatory properties, which makes it a promising candidate for countering inflammation-enhanced perinatal brain injury. In the present study we evaluated the effect of PRE-084 in a neonatal mouse model of inflammation-sensitized excitotoxic brain injury. From postnatal days 1 to 4, pups were pre-sensitized by intraperitoneal injections of IL-1beta (10ng). Two hours after the last IL-1beta dose, pups received an intracranial ibotenate injection, 1 hour after the insult they were randomly treated with i) 0.1 µg/g bodyweight PRE-084 or ii) vehicle. Administration of PRE-084 resulted in a significant decrease in cortical grey (mean length of the lesion: vehicle 780.00µm ± 495.35 vs. PRE-084 433.33µm±116.51; n=8–9; p<0.05) and adjacent white matter damage (mean length of the lesion: vehicle 767.50µm ± 489.07 vs. PRE-084 391.11µm±126.14; n=8–9; p<0.05). No sex-specific differences in lesion size were detected (n=3–6, p>0.05). PRE-084 treatment significantly reduced the number of isolectin B4-positive activated microglial cells in perilesional white matter (mean number of isolectin B4-positive activated microglia vehicle 36.40±6.96 vs. PRE-084 19.93±11.99; n=5; p<0.05). We are the first to report that PRE-084 reduces inflammation-sensitized NMDAR-mediated excitotoxic perinatal brain damage. Since sigma-1 receptor agonists are investigated in clinical trials in adult neurological diseases, they might be considered a promising therapeutic option also in perinatal brain injury.