Cacha Peeters-Scholte

Neuroprotection by Selective Nitric Oxide Synthase Inhibition at 24 Hours After Perinatal Hypoxia-Ischemia

Background and Purpose— Perinatal hypoxia-ischemia is a major cause of neonatal morbidity and mortality. Until now no established neuroprotective intervention after perinatal hypoxia-ischemia has been available. The delay in cell death after perinatal hypoxia-ischemia creates possibilities for therapeutic intervention after the initial insult. Excessive nitric oxide and reactive oxygen species generated on hypoxia-ischemia and reperfusion play a key role in the neurotoxic cascade. The present study examines the neuroprotective properties of neuronal and inducible but not endothelial nitric oxide synthase inhibition by 2-iminobiotin in a piglet model of perinatal hypoxia-ischemia. Methods— Twenty-three newborn piglets were subjected to 60 minutes of hypoxia-ischemia, followed by 24 hours of reperfusion and reoxygenation. Five additional piglets served as sham-operated controls. On reperfusion, piglets were randomly treated with either vehicle (n=12) or 2-iminobiotin (n=11). At 24 hours after hypoxia-ischemia, the cerebral energy state, presence of vasogenic edema, amount of apparently normal neuronal cells, caspase-3 activity, amount of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL)-positive cells, and degree of tyrosine nitration were assessed. Results— A 90% improvement in cerebral energy state, 90% reduction in vasogenic edema, and 60% to 80% reduction in apoptosis-related neuronal cell death were demonstrated in 2-iminobiotin-treated piglets at 24 hours after hypoxia- ischemia. A significant reduction in tyrosine nitration in the cerebral cortex was observed in 2-iminobiotin-treated piglets, indicating decreased formation of reactive nitrogen species. Conclusions— Simultaneous and selective inhibition of neuronal and inducible nitric oxide synthase by 2-iminobiotin is a promising strategy for neuroprotection after perinatal hypoxia-ischemia.

Effects of Allopurinol and Deferoxamine on Reperfusion Injury of the Brain in Newborn Piglets after Neonatal Hypoxia-Ischemia

The hypothesis was tested that treatment with allopurinol, a xanthine oxidase inhibitor, or deferoxamine, a chelator of nonprotein-bound iron, preserved cerebral energy metabolism, attenuated development of edema, and improved histologic outcome in the newborn piglet at 24 h after hypoxia-ischemia. Thirty-two newborn piglets were subjected to 1 h of hypoxia-ischemia by occluding both carotid arteries and reducing the fraction of inspired oxygen; five newborn piglets served as sham-operated controls. The depth of hypoxia-ischemia was controlled by phosphorous magnetic resonance spectroscopy. Upon reperfusion and reoxygenation, piglets received vehicle (n = 12), allopurinol (30 mg/kg/d, n = 10), or deferoxamine (12.5 mg/kg/d, n = 10). The cerebral energy status was determined with phosphorous magnetic resonance spectroscopy. The presence of vasogenic edema was assessed by T2-weighted magnetic resonance imaging. Brain cell injury was assessed with caspase-3 activity, histology, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end (TUNEL)-labeling. At 24 h after hypoxia-ischemia, the phosphocreatine/inorganic phosphate ratios were significantly decreased in vehicle-treated, but not in allopurinol- or deferoxamine-treated piglets. Water T2 values were significantly increased at 24 h after hypoxia-ischemia in cerebral cortex, thalamus, and striatum of vehicle-treated piglets, but not in allopurinol- and deferoxamine-treated piglets. No differences in caspase-3 activity, histologic outcome, or TUNEL-labeling were demonstrated between the three treatment groups. We suggest that allopurinol and deferoxamine may have an additional value in the treatment of perinatal hypoxia-ischemia with other neuroprotective agents or in combination with hypothermia.

Nitrosylation precedes caspase-3 activation and translocation of apoptosis-inducing factor in neonatal rat cerebral hypoxia-ischaemia

Excessive nitric oxide (NO) production after cerebral hypoxia‐ischaemia (HI) may induce cellular injury in various ways, including reaction with superoxide to form the highly reactive peroxynitrite. We characterized the spatial and temporal formation of peroxynitrite through immunohistochemical detection of nitrosylated proteins. Nitrotyrosine immunoreactivity peaked around 3 h post‐HI and was detected in areas of injury, as judged by the loss of microtubule‐associated protein‐2 (MAP‐2) staining, in neurones, glia and endothelial cells. Nitrotyrosine staining co‐localized with three other cellular markers of injury, active caspase‐3, nuclear translocation of apoptosis‐inducing factor (AIF) and an oligonucleotide hairpin probe detecting specific DNA strand breaks. The number of nitrotyrosine‐positive cells at early time points outnumbered the cells positive for the other three markers of injury, indicating that nitrosylation preceded caspase‐3 activation. Pharmacological inhibition of neuronal and inducible nitric oxide synthase (nNOS and iNOS) using 2‐iminobiotin, which has been demonstrated earlier to be neuroprotective, significantly reduced nitrotyrosine formation and caspase‐3 activation, but not nuclear translocation of AIF, in cortex and striatum of the ipsilatral hemisphere. In summary, nitrotyrosine is an early marker of cellular injury and inhibition of nNOS and iNOS is a promising strategy for neuroprotection after perinatal HI.

Long-term neuroprotection with 2-iminobiotin, an inhibitor of neuronal and inducible nitric oxide synthase, after cerebral hypoxia-ischemia in neonatal rats.

The short- and long-term neuroprotective effects of 2-iminobiotin, a selective inhibitor of neuronal and inducible nitric oxide synthase, were studied in 12-day-old rats following hypoxia-ischemia. Hypoxia-ischemia was induced by occlusion of the right carotid artery followed by 90 minutes of hypoxia (FiO2 0.08). Immediately on reoxygenation, 12 and 24 hours later the rats were treated with vehicle or 2-iminobiotin at a dose of 5.5, 10, 30, or 60 mg/kg per day. Histologic analysis of brain damage was performed at 6 weeks after hypoxia-ischemia. To assess early changes of cerebral tissue, levels of HSP70, nitrotyrosine, and cytochrome c were determined 24 hours after reoxygenation. Significant neuroprotection was obtained using a dose of 30 mg/kg per day of 2-iminobiotin. Levels of HSP70 were increased in the ipsilateral hemisphere in both groups (P<0.05), but the increase was significantly (P<0.05) less in the rats receiving the optimal dose of 2-iminobiotin (30 mg/kg per day). Hypoxia-ischemia did not lead to increased levels of nitrotyrosine, nor did 2-iminobiotin influence levels of nitrotyrosine. In contrast, hypoxia-ischemia induced an increase in cytochrome c level that was prevented by 2-iminobiotin. In conclusion, 2-iminobiotin administered after hypoxia-ischemia provides long-term neuroprotection. This neuroprotection is obtained by mechanisms other than a reduction of nitrotyrosine formation in proteins.

Inhibition of nNOS and iNOS following Hypoxia-Ischaemia Improves Long-Term Outcome but Does Not Influence the Inflammatory Response in the Neonatal Rat Brain

In this study, we tested the hypothesis that combined inhibition of nNOS and iNOS will reduce neuronal damage and the inflammatory response induced by perinatal hypoxia-ischaemia (HI). In 12-day-old rats, HI was induced by right carotid artery occlusion followed by 90 min of 8% O2. Immediately upon reoxygenation, the rats were treated with NOS inhibitors (n = 24) or placebo (n = 24). Neuropathology was scored at 6 weeks after HI on a 4-point scale (n = 12 per group). The expression of heat shock protein 70 (HSP70) and mRNA expression for cytokines were measured 12 h after HI (n = 12 per group). Histopathological analysis showed that the ipsilateral hemisphere in the NOS inhibition group was less damaged than in the placebo group (p < 0.05). HI induced a significant increase in HSP70 levels (p < 0.05) in the ipsilateral hemispheres, which tended to be lower in the NOS inhibition group (p = 0.07). HI induced an increase in mRNA expression for IL-1β, TNF-α and TNF-β, but there was no difference between the ipsi- and contralateral hemispheres. Combined inhibition of nNOS and iNOS did not induce any change in cytokine expression. We conclude that the long-term neuroprotective effects of combined nNOS and iNOS inhibition were not achieved by an altered cytokine response.

Effects of Selective Nitric Oxide Synthase Inhibition on IGF-1, Caspases and Cytokines in a Newborn Piglet Model of Perinatal Hypoxia-Ischaemia

Selective inhibition of neuronal and inducible nitric oxide synthase (NOS) with 2-iminobiotin previously showed a reduction in brain cell injury. In the present study, we investigated the effects of 2-iminobiotin treatment on insulin-like growth factor-1 (IGF-1) expression, caspase activity and cytokine expression in a newborn piglet model of perinatal hypoxia-ischaemia. Newborn piglets were subjected to 1 h of hypoxia-ischaemia and were treated intravenously with vehicle or 2-iminobiotin. Vehicle-treated piglets showed reduced IGF-1 mRNA expression and increased caspase-3 activity and DNA fragmentation. 2-Iminobiotin treatment, administered immediately upon reperfusion, prevented these observations. No differences in caspase-8 and -9 activity and cytokine [interleukin (IL)-1α/β, IL-6, tumour necrosis factor (TNF)-α, transforming growth factor (TGF)-β] mRNA expression were demonstrated between vehicle- and 2-iminobiotin-treated piglets at 24 h following hypoxia-ischaemia. IGF-1 mRNA correlated inversely with caspase-3 and transferase-mediated dUTP-biotin in situ nick end labelling score in the cortex, but positively with caspase-8. Cytokine mRNA did not correlate with IGF-1 mRNA, caspase-3 activity or DNA fragmentation. The present results indicate that the previously demonstrated neuroprotective effect of 2-iminobiotin treatment after perinatal hypoxia-ischaemia coincided with a preservation of the endogenous IGF-1 production and reduced caspase-3 activity, but not with a significant decrease in cytokine production.

Short-Term Dose–Response Characteristics of 2-Iminobiotin Immediately Postinsult in the Neonatal Piglet After Hypoxia-Ischemia

Background and Purpose— To determine the optimal dose of 2-iminobiotin (2-IB) for the treatment of moderate to severe asphyxia in a neonatal piglet model of hypoxia-ischemia. Methods— Newborn piglets were subjected to a 30-minute hypoxia-ischemia insult and randomly treated with vehicle or 2-IB (0.1 mg/kg, 0.2 mg/kg, or 1.0 mg/kg). aEEG background and seizure activity were scored after hypoxia-ischemia every 4 h until 24 h and at 48 h and neurobehavioral scores were obtained. Brain tissue was collected and processed for analysis of caspase-3 activity, histology, and tyrosine nitration. Results— A dose range of 0.1 to 1.0 mg/kg/dose of 2-IB improved short-term outcome as demonstrated by an increased survival with a normal aEEG and decreased nitrotyrosine staining in the 2-IB–treated animals, indicating decreased cellular damage. Neurobehavior, caspase-3 activity in thalamus, and histology scores were not significantly different. Conclusions— Based on survival with a normal aEEG, 0.2 mg/kg 2-IB is likely to be the most appropriate dose for use in future clinical trials in neonates with perinatal hypoxia-ischemia.

Short Term Safety and Pharmacokinetics of the Selective NOS Inhibitor 2-Iminobiotin in Asphyxiated Neonates during Therapeutic Hypothermia: Protocol for the 2-STEP Study

Background: Despite treatment with therapeutic hypothermia 45% of neonates with hypoxic-ischemic encephalopathy show an adverse outcome. Interventions combining hypothermia with additional neuroprotective strategies are needed in order to further improve the outcome of these neonates. The selective inhibitor of nNOS and iNOS 2-Iminobiotin has shown neuroprotective properties in animal models, but has not been combined with therapeutic hypothermia in humans. Methods: A single centre open label, prospective study will be performed. Term and near-term neonates treated with therapeutic hypothermia after perinatal asphyxia without major congenital malformations will be eligible for inclusion. In group A (N=6), patients will receive 4 bolus infusions of 2-iminobiotin (0.16 mg/kg) every 6 h. Short term safety will be assessed by monitoring of any changes in vital signs, aEEG or cerebral saturation using near-infrared spectrometry. For pharmacokinetic analyses, five blood samples of 0.4 ml per patient will be taken. Nonlinear mixed effects modelling (NONMEM) will be used to calculate individual area under the concentration time curves (AUC) as well as population parameters for clearance and distribution. After group A, an interim analysis will take place to determine the dosing regimen for group B (N=6). Discussion: This study will be the first to evaluate the use of 2-Iminobiotin in addition to therapeutic hypothermia in human neonates. Results from this study will contribute to the design and dosing regimen of a placebo controlled randomized controlled trial evaluating the efficacy of 2-Iminobiotin in this population as well as providing an understanding of 2-Iminobiotin pharmacokinetic parameters. Trail registration: European Clinical Trials Database (2014-004265-25) and the Netherlands Trial Register (www.trialregister.nl: NTR5221).