Systemic multipotent adult progenitor cells protect the cerebellum after asphyxia in fetal sheep.

Involvement of the cerebellum in the pathophysiology of hypoxic-ischemic encephalopathy (HIE) in preterm infants is increasingly recognized. We aimed to assess the neuroprotective potential of intravenously administered multipotent adult progenitor cells (MAPCs) in the preterm cerebellum. Instrumented preterm ovine fetuses were subjected to transient global hypoxia-ischemia (HI) by 25 minutes of umbilical cord occlusion at 0.7 of gestation. After reperfusion, two doses of MAPCs were administered intravenously. MAPCs are a plastic adherent bone-marrow-derived population of adult progenitor cells with neuroprotective potency in experimental and clinical studies. Global HI caused marked cortical injury in the cerebellum, histologically indicated by disruption of cortical strata, impeded Purkinje cell development, and decreased dendritic arborization. Furthermore, global HI induced histopathological microgliosis, hypomyelination, and disruption of white matter organization. MAPC treatment significantly prevented cortical injury and region-specifically attenuated white matter injury in the cerebellum following global HI. Diffusion tensor imaging (DTI) detected HI-induced injury and MAPC neuroprotection in the preterm cerebellum. This study has demonstrated in a preclinical large animal model that early systemic MAPC therapy improved structural injury of the preterm cerebellum following global HI. Microstructural improvement was detectable with DTI. These findings support the potential of MAPC therapy for the treatment of HIE and the added clinical value of DTI for the detection of cerebellar injury and the evaluation of cell-based therapy.

Preterm Brain Injury, Antenatal Triggers, and Therapeutics: Timing Is Key.

With a worldwide incidence of 15 million cases, preterm birth is a major contributor to neonatal mortality and morbidity, and concomitant social and economic burden Preterm infants are predisposed to life-long neurological disorders due to the immaturity of the brain. The risks are inversely proportional to maturity at birth. In the majority of extremely preterm infants (<28 weeks' gestation), perinatal brain injury is associated with exposure to multiple inflammatory perinatal triggers that include antenatal infection (i.e., chorioamnionitis), hypoxia-ischemia, and various postnatal injurious triggers (i.e., oxidative stress, sepsis, mechanical ventilation, hemodynamic instability). These perinatal insults cause a self-perpetuating cascade of peripheral and cerebral inflammation that plays a critical role in the etiology of diffuse white and grey matter injuries that underlies a spectrum of connectivity deficits in survivors from extremely preterm birth. This review focuses on chorioamnionitis and hypoxia-ischemia, which are two important antenatal risk factors for preterm brain injury, and highlights the latest insights on its pathophysiology, potential treatment, and future perspectives to narrow the translational gap between preclinical research and clinical applications.

Annexin A1 as Neuroprotective Determinant for Blood-Brain Barrier Integrity in Neonatal Hypoxic-Ischemic Encephalopathy.

Blood-brain barrier (BBB) disruption is associated with hypoxia-ischemia (HI) induced brain injury and life-long neurological pathologies. Treatment options are limited. Recently, we found that mesenchymal stem/stromal cell derived extracellular vesicles (MSC-EVs) protected the brain in ovine fetuses exposed to HI. We hypothesized that Annexin A1 (ANXA1), present in MSC-EVs, contributed to their therapeutic potential by targeting the ANXA1/Formyl peptide receptor (FPR), thereby preventing loss of the BBB integrity. Cerebral ANXA1 expression and leakage of albumin into the fetal ovine brain parenchyma after HI were analyzed by immunohistochemistry. For mechanistic insights, barrier integrity of primary fetal endothelial cells was assessed after oxygen-glucose deprivation (OGD) followed by treatment with MSC-EVs or human recombinant ANXA1 in the presence or absence of FPR inhibitors. Our study revealed that BBB integrity was compromised after HI which was improved by MSC-EVs containing ANXA1. Treatment with these MSC-EVs or ANXA1 improved BBB integrity after OGD, an effect abolished by FPR inhibitors. Furthermore, endogenous ANXA1 was depleted within 24 h after induction of HI in cerebovasculature and ependyma and upregulated 72 h after HI in microglia. Targeting ANXA1/FPR with ANXA1 in the immature brain has great potential in preventing BBB loss and concomitant brain injury following HI.

The influence of anesthetics on substantia nigra tyrosine hydroxylase expression and tau phosphorylation in the hypoxic-ischemic near-term lamb.

BackgroundGeneral anesthetics could protect key neurotransmitter systems, such as the dopaminergic system, from hypoxic-ischemic encephalopathy (HIE) by limiting excessive glutamatergic neurotransmission. However, anesthetics may adversely affect inflammation and tau phosphorylation.MethodsA near-term sheep model of HIE by umbilical cord occlusion (UCO) under anesthesia was used. The effect of propofol and isoflurane on the dopaminergic neurotransmitter phenotype in the substantia nigra (SN) was studied using tyrosine hydroxylase immunohistochemistry. The overall microglial response and tau phosphorylation were also measured in the SN, surrounding the midbrain gray matter structures and the hippocampal white matter.ResultsThe isoflurane-treated UCO group had fewer tyrosine hydroxylase-expressing neurons in the SN at 8 h after the insult than the propofol-treated UCO or sham-operated groups (P<0.05). The microglial response was unchanged in the SN region. In the thalamus and the hippocampal stratum moleculare layer, the propofol-treated UCO group had a lower microglial response than the corresponding sham-operated group. Both UCO and the use of anesthetics additively increased tau phosphorylation in the SN region, thalamus, and hippocampus.ConclusionThe choice of anesthetics is important for an emergency C-section. Propofol could potentially protect the dopaminergic neurotransmitter phenotype within the SN at the cost of a widespread increase in tau phosphorylation.

Chorioamnionitis, neuroinflammation, and injury: timing is key in the preterm ovine fetus.

BACKGROUND: Antenatal infection (i.e., chorioamnionitis) is an important risk factor for adverse neurodevelopmental outcomes after preterm birth. Destructive and developmental disturbances of the white matter are hallmarks of preterm brain injury. Understanding the temporal effects of antenatal infection in relation to the onset of neurological injury is crucial for the development of neurotherapeutics for preterm infants. However, these dynamics remain unstudied. METHODS: Time-mated ewes were intra-amniotically injected with lipopolysaccharide at 5, 12, or 24 h or 2, 4, 8, or 15 days before preterm delivery at 125 days gestational age (term ~ 150 days). Post mortem analyses for peripheral immune activation, neuroinflammation, and white matter/neuronal injury were performed. Moreover, considering the neuroprotective potential of erythropoietin (EPO) for perinatal brain injury, we evaluated (phosphorylated) EPO receptor (pEPOR) expression in the fetal brain following LPS exposure. RESULTS: Intra-amniotic exposure to this single bolus of LPS resulted in a biphasic systemic IL-6 and IL-8 response. In the developing brain, intra-amniotic LPS exposure induces a persistent microgliosis (IBA-1 immunoreactivity) but a shorter-lived increase in the pro-inflammatory marker COX-2. Cell death (caspase-3 immunoreactivity) was only observed when LPS exposure was greater than 8 days in the white matter, and there was a reduction in the number of (pre) oligodendrocytes (Olig2- and PDGFRα-positive cells) within the white matter at 15 days post LPS exposure only. pEPOR expression displayed a striking biphasic regulation following LPS exposure which may help explain contradicting results among clinical trials that tested EPO for the prevention of preterm brain injury. CONCLUSION: We provide increased understanding of the spatiotemporal pathophysiological changes in the preterm brain following intra-amniotic inflammation which may aid development of new interventions or implement interventions more effectively to prevent perinatal brain damage.

The Paradoxical Effects of Chronic Intra-Amniotic Ureaplasma parvum Exposure on Ovine Fetal Brain Development.

Chorioamnionitis is associated with adverse neurodevelopmental outcomes in preterm infants. Ureaplasma spp. are the microorganisms most frequently isolated from the amniotic fluid of women diagnosed with chorioamnionitis. However, controversy remains concerning the role of Ureaplasma spp. in the pathogenesis of neonatal brain injury. We hypothesize that reexposure to an inflammatory trigger during the perinatal period might be responsible for the variation in brain outcomes of preterms following Ureaplasma-driven chorioamnionitis. To investigate these clinical scenarios, we performed a detailed multimodal study in which ovine neurodevelopmental outcomes were assessed following chronic intra-amniotic Ureaplasma parvum (UP) infection either alone or combined with subsequent lipopolysaccharide (LPS) exposure. We show that chronic intra-amniotic UP exposure during the second trimester provoked a decrease in astrocytes, increased oligodendrocyte numbers, and elevated 5-methylcytosine levels. In contrast, short-term LPS exposure before preterm birth induced increased microglial activation, myelin loss, elevation of 5-hydroxymethylcytosine levels, and lipid profile changes. These LPS-induced changes were prevented by chronic preexposure to UP (preconditioning). These data indicate that chronic UP exposure has dual effects on preterm brain development in utero. On the one hand, prolonged UP exposure causes detrimental cerebral changes that may predispose to adverse postnatal clinical outcomes. On the other, chronic intra-amniotic UP exposure preconditions the brain against a second inflammatory hit. This study demonstrates that microbial interactions and the timing and duration of the inflammatory insults determine the effects on the fetal brain. Therefore, this study helps to understand the complex and diverse postnatal neurological outcomes following UP driven chorioamnionitis.

Neuroinflammation and structural injury of the fetal ovine brain following intra-amniotic Candida albicans exposure.

BACKGROUND: Intra-amniotic Candida albicans (C. Albicans) infection is associated with preterm birth and high morbidity and mortality rates. Survivors are prone to adverse neurodevelopmental outcomes. The mechanisms leading to these adverse neonatal brain outcomes remain largely unknown. To better understand the mechanisms underlying C. albicans-induced fetal brain injury, we studied immunological responses and structural changes of the fetal brain in a well-established translational ovine model of intra-amniotic C. albicans infection. In addition, we tested whether these potential adverse outcomes of the fetal brain were improved in utero by antifungal treatment with fluconazole. METHODS: Pregnant ewes received an intra-amniotic injection of 10(7) colony-forming units C. albicans or saline (controls) at 3 or 5 days before preterm delivery at 0.8 of gestation (term ~ 150 days). Fetal intra-amniotic/intra-peritoneal injections of fluconazole or saline (controls) were administered 2 days after C. albicans exposure. Post mortem analyses for fungal burden, peripheral immune activation, neuroinflammation, and white matter/neuronal injury were performed to determine the effects of intra-amniotic C. albicans and fluconazole treatment. RESULTS: Intra-amniotic exposure to C. albicans caused a severe systemic inflammatory response, illustrated by a robust increase of plasma interleukin-6 concentrations. Cerebrospinal fluid cultures were positive for C. albicans in the majority of the 3-day C. albicans-exposed animals whereas no positive cultures were present in the 5-day C. albicans-exposed and fluconazole-treated animals. Although C. albicans was not detected in the brain parenchyma, a neuroinflammatory response in the hippocampus and white matter was seen which was characterized by increased microglial and astrocyte activation. These neuroinflammatory changes were accompanied by structural white matter injury. Intra-amniotic fluconazole reduced fetal mortality but did not attenuate neuroinflammation and white matter injury. CONCLUSIONS: Intra-amniotic C. albicans exposure provoked acute systemic and neuroinflammatory responses with concomitant white matter injury. Fluconazole treatment prevented systemic inflammation without attenuating cerebral inflammation and injury.

Intra-Amniotic LPS Induced Region-Specific Changes in Presynaptic Bouton Densities in the Ovine Fetal Brain.

RATIONALE: Chorioamnionitis has been associated with increased risk for fetal brain damage. Although, it is now accepted that synaptic dysfunction might be responsible for functional deficits, synaptic densities/numbers after a fetal inflammatory challenge have not been studied in different regions yet. Therefore, we tested in this study the hypothesis that LPS-induced chorioamnionitis caused profound changes in synaptic densities in different regions of the fetal sheep brain. MATERIAL AND METHODS: Chorioamnionitis was induced by a 10 mg intra-amniotic LPS injection at two different exposure intervals. The fetal brain was studied at 125 days of gestation (term = 150 days) either 2 (LPS2D group) or 14 days (LPS14D group) after LPS or saline injection (control group). Synaptophysin immunohistochemistry was used to quantify the presynaptic density in layers 2-3 and 5-6 of the motor cortex, somatosensory cortex, entorhinal cortex, and piriforme cortex, in the nucleus caudatus and putamen and in CA1/2, CA3, and dentate gyrus of the hippocampus. RESULTS: There was a significant reduction in presynaptic bouton densities in layers 2-3 and 5-6 of the motor cortex and in layers 2-3 of the entorhinal and the somatosensory cortex, in the nucleus caudate and putamen and the CA1/2 and CA3 of the hippocampus in the LPS2D compared to control animals. Only in the motor cortex and putamen, the presynaptic density was significantly decreased in the LPS14 D compared to the control group. No changes were found in the dentate gyrus of the hippocampus and the piriforme cortex. CONCLUSION: We demonstrated that LPS-induced chorioamnionitis caused a decreased density in presynaptic boutons in different areas in the fetal brain. These synaptic changes seemed to be region-specific, with some regions being more affected than others, and seemed to be transient in some regions.