Alex Zwanenburg

Mesenchymal stem cells induce T-cell tolerance and protect the preterm brain after global hypoxia-ischemia.

Hypoxic-ischemic encephalopathy (HIE) in preterm infants is a severe disease for which no curative treatment is available. Cerebral inflammation and invasion of activated peripheral immune cells have been shown to play a pivotal role in the etiology of white matter injury, which is the clinical hallmark of HIE in preterm infants. The objective of this study was to assess the neuroprotective and anti-inflammatory effects of intravenously delivered mesenchymal stem cells (MSC) in an ovine model of HIE. In this translational animal model, global hypoxia-ischemia (HI) was induced in instrumented preterm sheep by transient umbilical cord occlusion, which closely mimics the clinical insult. Intravenous administration of 2 x 106 MSC/kg reduced microglial proliferation, diminished loss of oligodendrocytes and reduced demyelination, as determined by histology and Diffusion Tensor Imaging (DTI), in the preterm brain after global HI. These anti-inflammatory and neuroprotective effects of MSC were paralleled by reduced electrographic seizure activity in the ischemic preterm brain. Furthermore, we showed that MSC induced persistent peripheral T-cell tolerance in vivo and reduced invasion of T-cells into the preterm brain following global HI. These findings show in a preclinical animal model that intravenously administered MSC reduced cerebral inflammation, protected against white matter injury and established functional improvement in the preterm brain following global HI. Moreover, we provide evidence that induction of T-cell tolerance by MSC might play an important role in the neuroprotective effects of MSC in HIE. This is the first study to describe a marked neuroprotective effect of MSC in a translational animal model of HIE.

Cerebral inflammation and mobilization of the peripheral immune system following global hypoxia-ischemia in preterm sheep

BackgroundHypoxic-ischemic encephalopathy (HIE) is one of the most important causes of brain injury in preterm infants. Preterm HIE is predominantly caused by global hypoxia-ischemia (HI). In contrast, focal ischemia is most common in the adult brain and known to result in cerebral inflammation and activation of the peripheral immune system. These inflammatory responses are considered to play an important role in the adverse outcomes following brain ischemia. In this study, we hypothesize that cerebral and peripheral immune activation is also involved in preterm brain injury after global HI.MethodsPreterm instrumented fetal sheep were exposed to 25 minutes of umbilical cord occlusion (UCO) (n = 8) at 0.7 gestation. Sham-treated animals (n = 8) were used as a control group. Brain sections were stained for ionized calcium binding adaptor molecule 1 (IBA-1) to investigate microglial proliferation and activation. The peripheral immune system was studied by assessment of circulating white blood cell counts, cellular changes of the spleen and influx of peripheral immune cells (MPO-positive neutrophils) into the brain. Pre-oligodendrocytes (preOLs) and myelin basic protein (MBP) were detected to determine white matter injury. Electro-encephalography (EEG) was recorded to assess functional impairment by interburst interval (IBI) length analysis.ResultsGlobal HI resulted in profound activation and proliferation of microglia in the hippocampus, periventricular and subcortical white matter. In addition, non-preferential mobilization of white blood cells into the circulation was observed within 1 day after global HI and a significant influx of neutrophils into the brain was detected 7 days after the global HI insult. Furthermore, global HI resulted in marked involution of the spleen, which could not be explained by increased splenic apoptosis. In concordance with cerebral inflammation, global HI induced severe brain atrophy, region-specific preOL vulnerability, hypomyelination and persistent suppressed brain function.ConclusionsOur data provided evidence that global HI in preterm ovine fetuses resulted in profound cerebral inflammation and mobilization of the peripheral innate immune system. These inflammatory responses were paralleled by marked injury and functional loss of the preterm brain. Further understanding of the interplay between preterm brain inflammation and activation of the peripheral immune system following global HI will contribute to the development of future therapeutic interventions in preterm HIE.

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Protect the Fetal Brain After Hypoxia-Ischemia

Preterm neonates are susceptible to perinatal hypoxic‐ischemic brain injury, for which no treatment is available. In a preclinical animal model of hypoxic‐ischemic brain injury in ovine fetuses, we have demonstrated the neuroprotective potential of systemically administered mesenchymal stromal cells (MSCs). The mechanism of MSC treatment is unclear but suggested to be paracrine, through secretion of extracellular vesicles (EVs). Therefore, we investigated in this study the protective effects of mesenchymal stromal cell‐derived extracellular vesicles (MSC‐EVs) in a preclinical model of preterm hypoxic‐ischemic brain injury. Ovine fetuses were subjected to global hypoxia‐ischemia by transient umbilical cord occlusion, followed by in utero intravenous administration of MSC‐EVs. The therapeutic effects of MSC‐EV administration were assessed by analysis of electrophysiological parameters and histology of the brain. Systemic administration of MSC‐EVs improved brain function by reducing the total number and duration of seizures, and by preserving baroreceptor reflex sensitivity. These functional protections were accompanied by a tendency to prevent hypomyelination. Cerebral inflammation remained unaffected by the MSC‐EV treatment. Our data demonstrate that MSC‐EV treatment might provide a novel strategy to reduce the neurological sequelae following hypoxic‐ischemic injury of the preterm brain. Our study results suggest that a cell‐free preparation comprising neuroprotective MSC‐EVs could substitute MSCs in the treatment of preterm neonates with hypoxic‐ischemic brain injury, thereby circumventing the potential risks of systemic administration of living cells.

Systemic G-CSF attenuates cerebral inflammation and hypomyelination but does not reduce seizure burden in preterm sheep exposed to global hypoxia-ischemia.

Hypoxic-ischemic encephalopathy (HIE) is common in preterm infants, but currently no curative therapy is available. Cell-based therapy has a great potential in the treatment of hypoxic-ischemic preterm brain injury. Granulocyte-colony stimulating factor (G-CSF) is known to mobilize endogenous hematopoietic stem cells (HSC) and promotes proliferation of endogenous neural stem cells. On these grounds, we hypothesized that systemic G-CSF would be neuroprotective in a large translational animal model of hypoxic-ischemic injury in the preterm brain. Global hypoxia-ischemia (HI) was induced by transient umbilical cord occlusion in instrumented preterm sheep. G-CSF treatment (100μg/kg intravenously, during five consecutive days) was started one day before the global HI insult to ascertain mobilization of endogenous stem cells within the acute phase after global HI. Mobilization of HSC and neutrophils was studied by flow cytometry. Brain sections were stained for microglia (IBA-1), myelin basic protein (MBP) and myeloperoxidase (MPO) to study microglial proliferation, white matter injury and neutrophil invasion respectively. Electrographic seizure activity was analyzed using amplitude-integrated electroencephalogram (aEEG). G-CSF effectively mobilized CD34-positive HSC in the preterm sheep. In addition, G-CSF caused marked mobilization of neutrophils, but did not influence enhanced invasion of neutrophils into the preterm brain after global HI. Microglial proliferation and hypomyelination following global HI were reduced as a result of G-CSF treatment. G-CSF did not cause a reduction of the electrographic seizure activity after global HI. In conclusion, G-CSF induced mobilization of endogenous stem cells which was associated with modulation of the cerebral inflammatory response and reduced white matter injury in an ovine model of preterm brain injury after global HI. G-CSF treatment did not improve neuronal function as shown by seizure analysis. Our study shows that G-CSF treatment has neuroprotective potential following hypoxic-ischemic injury in the preterm brain.

Propofol administration to the maternal-fetal unit improved fetal EEG and influenced cerebral apoptotic pathway in preterm lambs suffering from severe asphyxia

BackgroundTerm and near-term infants are at high risk of developing brain injury and life-long disability if they have suffered from severe perinatal asphyxia. We hypothesized that propofol administration to the maternal-fetal unit can diminish cerebral injury in term and near-term infant fetuses in states of progressive severe asphyxia.MethodsForty-four late preterm lambs underwent total umbilical cord occlusion (UCO) or sham treatment in utero. UCO resulted in global asphyxia and cardiac arrest. After emergency cesarean section under either maternal propofol or isoflurane anesthesia, the fetuses were resuscitated and subsequently anesthetized the same way as their mothers.ResultsAsphyctic lambs receiving isoflurane showed a significant increase of total and low-frequency spectral power in bursts indicating seizure activity and more burst-suppression with a marked increase of interburst interval length during UCO. Asphyctic lambs receiving propofol showed less EEG changes. Propofol increased levels of anti-apoptotic B-cell lymphoma-extra large (Bcl-xL) and phosphorylated STAT-3 and reduced the release of cytochrome c from the mitochondria and the protein levels of activated cysteinyl aspartate-specific protease (caspase)-3, -9, and N-methyl-d-aspartate (NMDA) receptor.ConclusionsImprovement of fetal EEG during and after severe asphyxia could be achieved by propofol treatment of the ovine maternal-fetal unit. The underlying mechanism is probably the reduction of glutamate-induced cytotoxicity by down-regulation of NMDA receptors and an inhibition of the mitochondrial apoptotic pathway.

Automatic detection of burst synchrony in preterm infants

Electroencephalographic characteristics are useful in assessment of the functional status of specific neuronal connections relative to postmenstrual age. Interhemispheric burst synchrony (IBS) is a measure of the functional connectivity between the hemispheres in the maturing preterm brain. An algorithm was developed to assess IBS and was used in a prospective, longitudinal EEG study on 18 very preterm infants (< 32 weeks gestational age) with normal follow-up at 2 years of age. The preterm infants underwent weekly 4-hour multi-channel EEG recordings, resulting in n = 77 EEGs. After automated detection of bursts, the algorithm defines the start and end of interhemispheric synchronous burst activity, based on selection criteria found in literature. The algorithm was designed to emulate visual inspection, providing objective results in an automated manner. This approach may be applied in clinical use and open novel avenues to automated analysis in EEG monitoring and, moreover, it may facilitate assessment of the functional status of interhemispheric connections. As such, assessment of low interhemispheric synchrony may be associated with brain injury.

Comparison of ECG-based physiological markers for hypoxia in a preterm ovine model

Background:Current methods for assessing perinatal hypoxic conditions did not improve infant outcomes. Various waveform-based and interval-based ECG markers have been suggested, but not directly compared. We compare performance of ECG markers in a standardized ovine model for fetal hypoxia.Methods:Sixty-nine fetal sheep of 0.7 gestation had ECG recorded 4 h before, during, and 4 h after a 25-min period of umbilical cord occlusion (UCO), leading to severe hypoxia. Various ECG markers were calculated, among which were heart rate (HR), HR-corrected ventricular depolarization/repolarization interval (QTc), and ST-segment analysis (STAN) episodic and baseline rise markers, analogue to clinical STAN device alarms. Performance of interval- and waveform-based ECG markers was assessed by correlating predicted and actual hypoxic/normoxic state.Results:Of the markers studied, HR and QTc demonstrated high sensitivity (≥86%), specificity (≥96%), and positive predictive value (PPV) (≥86%) and detected hypoxia in ≥90% of fetuses at 4 min after UCO. In contrast, STAN episodic and baseline rise markers displayed low sensitivity (≤20%) and could not detect severe fetal hypoxia in 65 and 28% of the animals, respectively.Conclusion:Interval-based HR and QTc markers could assess the presence of severe hypoxia. Waveform-based STAN episodic and baseline rise markers were ineffective as markers for hypoxia.

ST waveform analysis for monitoring hypoxic distress in fetal sheep after prolonged umbilical cord occlusion

Introduction The inconclusive clinical results for ST-waveform analysis (STAN) in detecting fetal hypoxemia may be caused by the signal processing of the STAN-device itself. We assessed the performance of a clinical STAN device in signal processing and in detecting hypoxemia in a fetal sheep model exposed to prolonged umbilical cord occlusion (UCO). Methods Eight fetal lambs were exposed to 25 minutes of UCO. ECG recordings were analyzed during a baseline period and during UCO. STAN-event rates and timing of episodic T/QRS rise, baseline T/QRS rise and the occurrence of biphasic ST-waveforms, as well as signal loss, were assessed. Results During baseline conditions of normoxemia, a median of 40 (IQR, 25–70) STAN-events per minute were detected, compared to 10 (IQR, 2–22) during UCO. During UCO STAN-events were detected in five subjects within 10 minutes and in six subjects after 18 minutes, respectively. Two subjects did not generate any STAN-event during UCO. Biphasic ST event rate was reduced during UCO (median 0, IQR 0–5), compared to baseline (median 32, IQR, 6–55). ST-waveforms could not be assessed in 62% of the recording time during UCO, despite a good quality of the ECG signal. Conclusions The STAN device showed limitations in detecting hypoxemia in fetal sheep after prolonged UCO. The STAN device produced high false positive event rates during baseline and did not detect T/QRS changes adequately after prolonged fetal hypoxemia. During 14% of baseline and 62% of the UCO period, the STAN-device could not process the ECG signal, despite its good quality. Resolving these issues may improve the clinical performance of the STAN device.