Anna-Lena Leverin

Bacterial endotoxin sensitizes the immature brain to hypoxic–ischaemic injury

Epidemiological studies show a markedly increased risk of cerebral palsy following the combined exposure of infection and birth asphyxia. However, the underlying mechanisms of this increased vulnerability remain unclear. We have examined the effects of a low dose of bacterial endotoxin on hypoxic–ischaemic injury in the immature brain of rats. Bacterial endotoxin (lipopolysaccharide 0.3 mg/kg) was administered to 7‐day‐old rats 4 h prior to unilateral hypoxia–ischaemia and the neurological outcome was determined 3 days later. Rectal temperature and cerebral blood flow was measured during the study and the expression of CD14 and toll‐like receptor‐4 mRNA in the brain was examined. We found that a low dose of endotoxin dramatically sensitizes the immature brain to injury and induces cerebral infarction in response to short periods of hypoxia–ischaemia that by themselves caused no or little injury. This effect could not be explained by a reduction in cerebral blood flow or hyperthermia. In association with the sensitization of injury we found an altered expression of CD14 mRNA and toll‐like receptor‐4 mRNA in the brain. These results suggest that the innate immune system may be involved in the vulnerability of the immature brain following the combination of infection and hypoxia–ischaemia.

IGF-I neuroprotection in the immature brain after hypoxia-ischemia, involvement of Akt and GSK3β?

Insulin‐like growth factor I (IGF‐I) is a neurotrophic factor that promotes neuronal growth, differentiation and survival. Neuroprotective effects of IGF‐I have previously been shown in adult and juvenile rat models of brain injury. We wanted to investigate the neuroprotective effect of IGF‐I after hypoxia‐ischemia (HI) in 7‐day‐old neonatal rats and the mechanisms of IGF‐I actions in vivo. We also wanted to study effects of HI and/or IGF‐I on the serine/threonine kinases Akt and glycogen synthase kinase 3β (GSK3β) in the phophatidylinositol‐3 kinase (PI3K) pathway. Immediately after HI, phosphorylated Akt (pAkt) and phosphorylated GSK3β (pGSK3β) immunoreactivity was lost in the ipsilateral and reduced in the contralateral hemisphere. After 45 min, pAkt levels were restored to control values, whereas pGSK3β remained low 4 h after HI. Administration of IGF‐I (50 µg i.c.v.) after HI resulted in a 40% reduction in brain damage (loss of microtubule‐associated protein) compared with vehicle‐treated animals. IGF‐I treatment without HI was shown to increase pAkt whereas pGSK3β decreased in the cytosol, but increased in the nuclear fraction. IGF‐I treatment after HI increased pAkt in the cytosol and pGSK3β in both the cytosol and the nuclear fraction in the ipsilateral hemisphere compared with vehicle‐treated rats, concomitant with a reduced caspase‐3‐ and caspase‐9‐like activity. In conclusion, IGF‐I induces activation of Akt during recovery after HI which, in combination with inactivation of GSK3β, may explain the attenuated activation of caspases and reduction of injury in the immature brain.

Effect of lipopolysaccharide on global gene expression in the immature rat brain

To improve the understanding of the molecular mechanisms whereby lipopolysaccharide (LPS) affects the immature brain, global gene expression following LPS exposure was investigated in neonatal rats. Brains (n = 5/time point) were sampled 2, 6, and 72 h after LPS and compared with age-matched controls. The mRNA from each brain was analyzed separately on Affymextrix GeneChip Rat Expression Set 230. The number of genes regulated after LPS were 847 at 2 h, 1564 at 6 h, and 1546 genes at 72 h. Gene ontology analysis demonstrated that, at both 2 and 6 h after LPS, genes associated with protein metabolism, response to external stimuli and stress (immune and inflammatory response, chemotaxis) and cell death were overrepresented. At 72 h, the most strongly regulated genes belonged to secretion of neurotransmitters, transport, synaptic transmission, cell migration, and neurogenesis. Several pathways associated with cell death/survival were identified (caspase-tumor necrosis factor α [TNF-α]-, p53-, and Akt/phosphatidylinositol-3-kinase (PI3 K)–dependent mechanisms). Caspase-3 activity increased and phosphorylation of Akt decreased 8 h after peripheral LPS exposure. These results show a complex cerebral response to peripheral LPS exposure. In addition to the inflammatory response, a significant number of cell death-associated genes were identified, which may contribute to increased vulnerability of the immature brain to hypoxia-ischemia (HI) following LPS exposure.

Adenosine A1 receptor agonism in the immature rat brain and heart

We examined if the adenosine A1 receptor agonist adenosine amine congener (ADAC, 100 μg/kg i.p.) is neuroprotective in 7-day-old rats subjected to hypoxic ischemia. Brain damage, evaluated as weight deficit and gross morphology, was not affected by ADAC treatment. Nonetheless, ADAC (100 μg/kg i.p.) reduced heart rate by 44% (p<0.0001), indicating that the dose given was pharmacologically active. Adenosine A1 receptors were determined by [3H] 1,3-dipropyl-8-cyclopentylxanthine (DPCPX)-binding and levels were 23% of the adult levels. GTP did not affect [3H] DPCPX-binding in the cerebral cortex at postnatal day 7 whereas there was strong enhancement of [3H] DPCPX-binding in the heart. This suggested a poor G-protein coupling at postnatal day 7 in the brain, which also was confirmed using GTP [γ-35S]-binding in the presence of an adenosine A1 receptor agonist. Thus, the lack of a neuroprotective effect of ADAC may be explained by the fact that adenosine A1 receptors are not part of a functional unit in the 7-day-old rat brain.

Brain Barrier Properties and Cerebral Blood Flow in Neonatal Mice Exposed to Cerebral Hypoxia-Ischemia

Insults to the developing brain often result in irreparable damage resulting in long-term deficits in motor and cognitive functions. The only treatment today for hypoxic-ischemic encephalopathy (HIE) in newborns is hypothermia, which has limited clinical benefit. We have studied changes to the blood–brain barriers (BBB) as well as regional cerebral blood flow (rCBF) in a neonatal model of HIE to further understand the underlying pathologic mechanisms. Nine-day old mice pups, brain roughly equivalent to the near-term human fetus, were subjected to hypoxia-ischemia. Hypoxia-ischemia increased BBB permeability to small and large molecules within hours after the insult, which normalized in the following days. The opening of the BBB was associated with changes to BBB protein expression whereas gene transcript levels were increased showing direct molecular damage to the BBB but also suggesting compensatory mechanisms. Brain pathology was closely related to reductions in rCBF during the hypoxia as well as the areas with compromised BBB showing that these are intimately linked. The transient opening of the BBB after the insult is likely to contribute to the pathology but at the same time provides an opportunity for therapeutics to better reach the infarcted areas in the brain.

Isolation of brain mitochondria from neonatal mice.

J. Neurochem. (2011) 10.1111/j.1471‐4159.2011.07525.x

Magnesium induces preconditioning of the neonatal brain via profound mitochondrial protection

Magnesium sulphate (MgSO4) given to women in preterm labor reduces cerebral palsy in their offspring but the mechanism behind this protection is unclear, limiting its effective, safe clinical implementation. Previous studies suggest that MgSO4 is not neuroprotective if administered during or after the insult, so we hypothesised that MgSO4 induces preconditioning in the immature brain. Therefore, we administered MgSO4 at various time-points before/after unilateral hypoxia-ischemia (HI) in seven-day-old rats. We found that MgSO4 treatment administered as a bolus between 6 days and 12 h prior to HI markedly reduced the brain injury, with maximal protection achieved by 1.1 mg/g MgSO4 administered 24 h before HI. As serum magnesium levels returned to baseline before the induction of HI, we ascribed this reduction in brain injury to preconditioning. Cerebral blood flow was unaffected, but mRNAs/miRNAs involved in mitochondrial function and metabolism were modulated by MgSO4. Metabolomic analysis (H+-NMR) disclosed that MgSO4 attenuated HI-induced increases in succinate and prevented depletion of high-energy phosphates. MgSO4 pretreatment preserved mitochondrial respiration, reducing ROS production and inflammation after HI. Therefore, we propose that MgSO4 evokes preconditioning via induction of mitochondrial resistance and attenuation of inflammation.

Magnesium sulphate induces preconditioning in preterm rodent models of cerebral hypoxia-ischemia

Brain injury in preterm infants represents a substantial clinical problem associated with development of motor impairment, cognitive deficits and psychiatric problems. According to clinical studies, magnesium sulphate (MgSO4) given to women in preterm labor reduces the risk of cerebral palsy in the offspring but the mechanisms behind its neuroprotective effects are still unclear. Our aim was to explore whether MgSO4 induces tolerance (preconditioning) in the preterm rodent brain. For this purpose we established a model of perinatal hypoxia‐ischemia (HI) in postnatal day 4 rats and also applied a recently developed postnatal day 5 mouse model of perinatal brain injury.

Neuroprotective exendin-4 enhances hypothermia therapy in a model of hypoxic-ischaemic encephalopathy

Hypoxic-ischaemic encephalopathy (HIE) causes 25% of neonatal deaths worldwide. Rocha-Ferreira et al. demonstrate that a diabetes drug protects the neonatal brain in a model of acute HIE, and confirm that the required receptor is found in human perinatal brain tissue. Synergistic combination with clinical hypothermia enhances therapy, supporting potential translation.