The effect of levomepromazine on the healthy and injured developing mouse brain - An in vitro and in vivo study.

Levomepromazine (LMP) is a phenothiazine neuroleptic drug with strong analgesic and sedative properties that is increasingly used off-label in pediatrics and is being discussed as an adjunct therapy in neonatal intensive care. Basic research points towards neuroprotective potential of phenothiazines, but LMP's effect on the developing brain is currently unknown. The aim of the present study was to assess LMP as a pharmacologic strategy in established neonatal in vitro and in vivo models of the healthy and injured developing mouse brain. In vitro, HT-22 cells kept exposure-naïve or injured by glutamate were pre-treated with vehicle or increasing doses of LMP and cell viability was determined. In vivo, LMP's effects were first assessed in 5-day-old healthy, uninjured CD-1 mouse pups receiving a single intraperitoneal injection of vehicle or different dosages of LMP. In a second step, mouse pups were subjected to excitotoxic brain injury and subsequently treated with vehicle or LMP. Endpoints included somatometric data as well as histological and immunohistochemical analyses. In vitro, cell viability in exposure-naïve cells was significantly reduced by high doses of LMP, but remained unaffected in glutamate-injured cells. In vivo, no specific toxic effects of LMP were observed neither in healthy mouse pups nor in experimental animals subjected to excitotoxic injury, but body weight gain was significantly lower following higher-dose LMP treatment. Also, LMP failed to produce a neuroprotective effect in the injured developing brain. Additional studies are required prior to a routine clinical use of LMP in neonatal intensive care units.

Short-, Mid-, and Long-Term Effect of Granulocyte Colony-Stimulating Factor/Stem Cell Factor and Fms-Related Tyrosine Kinase 3 Ligand Evaluated in an In Vivo Model of Hypoxic-Hyperoxic Ischemic Neonatal Brain Injury.

Hematopoietic growth factors are considered to bear neuroprotective potential. We have previously shown that delayed treatment with granulocyte colony-stimulating factor (G-CSF)/stem cell factor (SCF) and Fms-related tyrosine kinase 3 ligand (FL) ameliorates excitotoxic neonatal brain injury. The effect of these substances in combined-stressor neonatal brain injury models more closely mimicking clinical conditions has not been investigated. The aim of this study was to assess the short-, mid-, and long-term neuroprotective potential of G-CSF/SCF and FL in a neonatal model of hypoxic-hyperoxic ischemic brain injury. Five-day-old (P5) CD-1 mice were subjected to unilateral common carotid artery ligation and subsequent alternating periods of hypoxia and hyperoxia for 65 minutes. Sixty hours after injury, pups were randomly assigned to intraperitoneal treatment with (i) G-CSF (200 µg/kg)/SCF (50 µg/kg), (ii) FL (100 µg/kg), or (iii) vehicle every 24 hours for three or five consecutive days. Histopathological and functional outcomes were evaluated on P10, P18, and P90. Baseline outcome parameters were established in sham-treated and healthy control animals. Gross brain injury did not significantly differ between treatment groups at any time point. On P10, caspase-3 activation and caspase-independent apoptosis were similar between treatment groups; cell proliferation and the number of BrdU-positive vessels did not differ on P18 or P90. Neurobehavioral assessment did not reveal significant differences between treatment groups in accelerod performance, open field behavior, or novel object recognition capacity on P90. Turning behavior was more frequently observed in G-CSF/SCF- and FL-treated animals. No sex-specific differences were detected in any outcome parameter evaluated. In hypoxic-hyperoxic ischemic neonatal brain injury, G-CSF/SCF and FL treatment does not convey neuroprotection. Prior to potential clinical use, meticulous assessment of these hematopoietic growth factors is mandated.

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.

ALG8-CDG: novel patients and review of the literature.

BACKGROUND: Since 1980, about 100 types of congenital disorders of glycosylation (CDG) have been reported representing an expanding group of inherited disorders. ALG8-CDG (= CDG-Ih) is one of the less frequently reported types of CDG, maybe due to its severe multi-organ involvement with coagulation disturbances, edema, massive gastrointestinal protein loosing enteropathy, cataracts, and often early death. We report three additional patients, provide an update on two previously reported, and summarize features of ten patients reported in literature. RESULTS: Of 15 ALG8-CDG patients, three were homozygous and 12 compound heterozygous. There were multiple prenatal abnormalities in 6/12 patients. In 13/15, there were symptoms at birth, 9/15 died within 12 months. Birth weight was appropriate in 11/12, only one was small for gestational age. Prematurity was reported in 7/12. Hydrops fetalis was noticed in 3, edemas in 11/13; gastrointestinal symptoms in 9/14; structural brain pathology, psychomental retardation, seizures, ataxia in 12/13, muscle hypotonia in 13/14. Common dysmorphic signs were: low set ears, macroglossia, hypertelorism, pes equinovarus, campto- and brachydactyly (13/15). In 10/11, there was coagulopathy, in 8/11 elevated transaminases; thrombocytopenia was present in 9/9. Eye involvement was reported in 9/14. CDG typical skin involvement was reported in 8/13. CONCLUSION: In ALG8-CDG, isoelectric focusing of transferrin in serum or plasma shows an abnormal sialotransferrin pattern. The diagnosis is confirmed by mutation analysis in ALG8; all patients reported so far had point mutations or small deletions. The prognosis is generally poor. Thus, a timely and correct diagnosis is important for counselling.

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.

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.

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.