Eridan Rocha-Ferreira

Brain Cell Death Is Reduced With Cooling by 3.5°C to 5°C but Increased With Cooling by 8.5°C in a Piglet Asphyxia Model

Background and Purpose— In infants with moderate to severe neonatal encephalopathy, whole-body cooling at 33°C to 34°C for 72 hours is standard care with a number needed to treat to prevent a adverse outcome of 6 to 7. The precise brain temperature providing optimal neuroprotection is unknown. Methods— After a quantified global cerebral hypoxic-ischemic insult, 28 piglets aged <24 hours were randomized (each group, n=7) to (1) normothermia (38.5°C throughout) or whole-body cooling 2 to 26 hours after insult to (2) 35°C, (3) 33.5°C, or (4) 30°C. At 48 hours after hypoxia-ischemia, delayed cell death (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling and cleaved caspase 3) and microglial ramification (ionized calcium-binding adapter molecule 1) were evaluated. Results— At 48 hours after hypoxia-ischemia, substantial cerebral injury was found in the normothermia and 30°C hypothermia groups. However, with 35°C and 33.5°C cooling, a clear reduction in delayed cell death and microglial activation was observed in most brain regions (P<0.05), with no differences between 35°C and 33.5°C cooling groups. A protective pattern was observed, with U-shaped temperature dependence in delayed cell death in periventricular white matter, caudate nucleus, putamen, hippocampus, and thalamus. A microglial activation pattern was also seen, with inverted U-shaped temperature dependence in periventricular white matter, caudate nucleus, internal capsule, and hippocampus (all P<0.05). Conclusions— Cooling to 35°C (an absolute drop of 3.5°C as in therapeutic hypothermia protocols) or to 33.5°C provided protection in most brain regions after a cerebral hypoxic-ischemic insult in the newborn piglet. Although the relatively wide therapeutic range of a 3.5°C to 5°C drop in temperature reassured, overcooling (an 8.5°C drop) was clearly detrimental in some brain regions.

Neuronal c-Jun is required for successful axonal regeneration, but the effects of phosphorylation of its N-terminus are moderate.

J. Neurochem. (2012) 121, 607–618.

Plasticity in the Neonatal Brain following Hypoxic-Ischaemic Injury

Hypoxic-ischaemic damage to the developing brain is a leading cause of child death, with high mortality and morbidity, including cerebral palsy, epilepsy, and cognitive disabilities. The developmental stage of the brain and the severity of the insult influence the selective regional vulnerability and the subsequent clinical manifestations. The increased susceptibility to hypoxia-ischaemia (HI) of periventricular white matter in preterm infants predisposes the immature brain to motor, cognitive, and sensory deficits, with cognitive impairment associated with earlier gestational age. In term infants HI causes selective damage to sensorimotor cortex, basal ganglia, thalamus, and brain stem. Even though the immature brain is more malleable to external stimuli compared to the adult one, a hypoxic-ischaemic event to the neonate interrupts the shaping of central motor pathways and can affect normal developmental plasticity through altering neurotransmission, changes in cellular signalling, neural connectivity and function, wrong targeted innervation, and interruption of developmental apoptosis. Models of neonatal HI demonstrate three morphologically different types of cell death, that is, apoptosis, necrosis, and autophagy, which crosstalk and can exist as a continuum in the same cell. In the present review we discuss the mechanisms of HI injury to the immature brain and the way they affect plasticity.

Peptidylarginine deiminases: novel drug targets for prevention of neuronal damage following hypoxic ischemic insult (HI) in neonates

Neonatal hypoxic ischaemic (HI) injury frequently causes neural impairment in surviving infants. Our knowledge of the underlying molecular mechanisms is still limited. Protein deimination is a post‐translational modification caused by Ca+2‐regulated peptidylarginine deiminases (PADs), a group of five isozymes that display tissue‐specific expression and different preference for target proteins. Protein deimination results in altered protein conformation and function of target proteins, and is associated with neurodegenerative diseases, gene regulation and autoimmunity. In this study, we used the neonatal HI and HI/infection [lipopolysaccharide (LPS) stimulation] murine models to investigate changes in protein deimination. Brains showed increases in deiminated proteins, cell death, activated microglia and neuronal loss in affected brain areas at 48 h after hypoxic ischaemic insult. Upon treatment with the pan‐PAD inhibitor Cl‐amidine, a significant reduction was seen in microglial activation, cell death and infarct size compared with control saline or LPS‐treated animals. Deimination of histone 3, a target protein of the PAD4 isozyme, was increased in hippocampus and cortex specifically upon LPS stimulation and markedly reduced following Cl‐amidine treatment. Here, we demonstrate a novel role for PAD enzymes in neural impairment in neonatal HI Encephalopathy, highlighting their role as promising new candidates for drug‐directed intervention in neurotrauma.

Antimicrobial peptides and complement in neonatal hypoxia-ischemia induced brain damage.

Hypoxic-ischemic encephalopathy (HIE) is a clinical condition in the neonate, resulting from oxygen deprivation around the time of birth. HIE affects 1–5/1000 live births worldwide and is associated with the development of neurological deficits, including cerebral palsy, epilepsy, and cognitive disabilities. Even though the brain is considered as an immune-privileged site, it has innate and adaptive immune response and can produce complement (C) components and antimicrobial peptides (AMPs). Dysregulation of cerebral expression of AMPs and C can exacerbate or ameliorate the inflammatory response within the brain. Brain ischemia triggers a prolonged inflammatory response affecting the progression of injury and secondary energy failure and involves both innate and adaptive immune systems, including immune-competent and non-competent cells. Following injury to the central nervous system (CNS), including neonatal hypoxia-ischemia (HI), resident microglia, and astroglia are the main cells providing immune defense to the brain in a stimulus-dependent manner. They can express and secrete pro-inflammatory cytokines and therefore trigger prolonged inflammation, resulting in neurodegeneration. Microglial cells express and release a wide range of inflammation-associated molecules including several components of the complement system. Complement activation following neonatal HI injury has been reported to contribute to neurodegeneration. Astrocytes can significantly affect the immune response of the CNS under pathological conditions through production and release of pro-inflammatory cytokines and immunomodulatory AMPs. Astrocytes express β-defensins, which can chemoattract and promote maturation of dendritic cells (DC), and can also limit inflammation by controlling the viability of these same DC. This review will focus on the balance of complement components and AMPs within the CNS following neonatal HI injury and the effect of that balance on the subsequent brain damage.

Inhibition of Signal Transducer and Activator of Transcription 3 (STAT3) reduces neonatal hypoxic‐ischaemic brain damage

Hypoxic‐ischaemic encephalopathy is a leading cause of child death, with high mortality and morbidity, including cerebral palsy, epilepsy and cognitive disabilities. Hypoxia‐ischaemia (HI) strongly up‐regulates Signal Transducer and Activator of Transcription 3 (STAT3) in the immature brain. Our aim was to establish whether STAT3 up‐regulation is associated with neonatal HI‐brain damage and evaluate the phosphorylated STAT3‐contribution from different cell types in eliciting damage. We subjected postnatal day seven mice to unilateral carotid artery ligation followed by 60 min hypoxia. Neuronal STAT3‐deletion reduced cell death, tissue loss, microglial and astroglial activation in all brain regions. Astroglia‐specific STAT3‐deletion also reduced cell death, tissue loss and microglial activation, although not as strongly as the deletion in neurons. Systemic pre‐insult STAT3‐blockade at tyrosine 705 (Y705) with JAK2‐inhibitor WP1066 reduced microglial and astroglial activation to a more moderate degree, but in a pattern similar to the one produced by the cell‐specific deletions. Our results suggest that STAT3 is a crucial factor in neonatal HI‐brain damage and its removal in neurons or astrocytes, and, to some extent, inhibition of its phosphorylation via JAK2‐blockade reduces inflammation and tissue loss. Overall, the protective effects of STAT3 inactivation make it a possible target for a therapeutic strategy in neonatal HI.

Inhaled 45–50% argon augments hypothermic brain protection in a piglet model of perinatal asphyxia

Cooling to 33.5 °C in babies with neonatal encephalopathy significantly reduces death and disability, however additional therapies are needed to maximize brain protection. Following hypoxia–ischemia we assessed whether inhaled 45–50% Argon from 2–26 h augmented hypothermia neuroprotection in a neonatal piglet model, using MRS and aEEG, which predict outcome in babies with neonatal encephalopathy, and immunohistochemistry. Following cerebral hypoxia–ischemia, 20 Newborn male Large White piglets < 40 h were randomized to: (i) Cooling (33 °C) from 2–26 h (n = 10); or (ii) Cooling and inhaled 45–50% Argon (Cooling + Argon) from 2–26 h (n = 8). Whole-brain phosphorus-31 and regional proton MRS were acquired at baseline, 24 and 48 h after hypoxia–ischemia. EEG was monitored. At 48 h after hypoxia–ischemia, cell death (TUNEL) was evaluated over 7 brain regions. There were no differences in body weight, duration of hypoxia–ischemia or insult severity; throughout the study there were no differences in heart rate, arterial blood pressure, blood biochemistry and inotrope support. Two piglets in the Cooling + Argon group were excluded. Comparing Cooling + Argon with Cooling there was preservation of whole-brain MRS ATP and PCr/Pi at 48 h after hypoxia–ischemia (p < 0.001 for both) and lower 1H MRS lactate/N acetyl aspartate in white (p = 0.03 and 0.04) but not gray matter at 24 and 48 h. EEG background recovery was faster (p < 0.01) with Cooling + Argon. An overall difference between average cell-death of Cooling versus Cooling + Argon was observed (p < 0.01); estimated cells per mm2 were 23.9 points lower (95% C.I. 7.3–40.5) for the Cooling + Argon versus Cooling. Inhaled 45–50% Argon from 2–26 h augmented hypothermic protection at 48 h after hypoxia–ischemia shown by improved brain energy metabolism on MRS, faster EEG recovery and reduced cell death on TUNEL. Argon may provide a cheap and practical therapy to augment cooling for neonatal encephalopathy.

The role of different strain backgrounds in bacterial endotoxin-mediated sensitization to neonatal hypoxic-ischemic brain damage.

Graphical abstract

Immediate remote ischemic postconditioning after hypoxia ischemia in piglets protects cerebral white matter but not grey matter

Remote ischemic postconditioning (RIPostC) is a promising therapeutic intervention whereby brief episodes of ischemia/reperfusion of one organ (limb) mitigate damage in another organ (brain) that has experienced severe hypoxia-ischemia. Our aim was to assess whether RIPostC is protective following cerebral hypoxia-ischemia in a piglet model of neonatal encephalopathy (NE) using magnetic resonance spectroscopy (MRS) biomarkers and immunohistochemistry. After hypoxia-ischemia (HI), 16 Large White female newborn piglets were randomized to: (i) no intervention (n = 8); (ii) RIPostC – with four, 10-min cycles of bilateral lower limb ischemia/reperfusion immediately after HI (n = 8). RIPostC reduced the hypoxic-ischemic-induced increase in white matter proton MRS lactate/N acetyl aspartate (p = 0.005) and increased whole brain phosphorus-31 MRS ATP (p = 0.039) over the 48 h after HI. Cell death was reduced with RIPostC in the periventricular white matter (p = 0.03), internal capsule (p = 0.002) and corpus callosum (p = 0.021); there was reduced microglial activation in corpus callosum (p = 0.001) and more surviving oligodendrocytes in corpus callosum (p = 0.029) and periventricular white matter (p = 0.001). Changes in gene expression were detected in the white matter at 48 h, including KATP channel and endothelin A receptor. Immediate RIPostC is a potentially safe and promising brain protective therapy for babies with NE with protection in white but not grey matter.

Immediate Remote Ischemic Postconditioning Reduces Brain Nitrotyrosine Formation in a Piglet Asphyxia Model

Remote ischemic postconditioning (RIPostC) is a promising therapeutic intervention that could be administered as an alternative to cooling in cases of perinatal hypoxia-ischemia (HI). In the current study we hypothesized that RIPostC in the piglet model of birth asphyxia confers protection by reducing nitrosative stress and subsequent nitrotyrosine formation, as well as having an effect on glial immunoreactivity. Postnatal day 1 (P1) piglets underwent HI brain injury and were randomised to HI (control) or HI + RIPostC. Immunohistochemistry assessment 48 hours after HI revealed a significant decrease in brain nitrotyrosine deposits in the RIPostC-treated group (p = 0.02). This was accompanied by a significant increase in eNOS expression (p < 0.0001) and decrease in iNOS (p = 0.010), with no alteration in nNOS activity. Interestingly, RIPostC treatment was associated with a significant increase in GFAP (p = 0.002) and IBA1 (p = 0.006), markers of astroglial and microglial activity, respectively. The current study demonstrates a beneficial effect of RIPostC therapy in the preclinical piglet model of neonatal asphyxia, which appears to be mediated by modulation of nitrosative stress, despite glial activation.