CXCL10 is a crucial chemoattractant for efficient intranasal delivery of mesenchymal stem cells to the neonatal hypoxic-ischemic brain.

BACKGROUND: Hypoxic-Ischemic Encephalopathy (HIE) is a leading cause of mortality and morbidity in newborns. Recent research has shown promise in using intranasal mesenchymal stem cell (MSC) therapy if administered within 10 days after Hypoxia-Ischemia (HI) in neonatal mice. MSCs migrate from the nasal cavity to the cerebral lesion in response to chemotactic cues. Which exact chemokines are crucial for MSC guidance to the HI lesion is currently not fully understood. This study investigates the role of CXCL10 in MSC migration towards the HI-injured brain. METHODS: HI was induced in male and female 9-day-old C57BL/6 mice followed by intranasal MSC treatment at day 10 or 17 post-HI. CXCL10 protein levels, PKH26-labeled MSCs and lesion size were assessed by ELISA, immunofluorescent imaging and MAP2 staining respectively. At day 17 post-HI, when CXCL10 levels were reduced, intracranial CXCL10 injection and intranasal PKH26-labeled MSC administration were combined to assess CXCL10-guided MSC migration. MSC treatment efficacy was evaluated after 18 days, measuring lesion size, motor outcome (cylinder rearing task), glial scarring (GFAP staining) and neuronal density (NeuN staining) around the lesion. Expression of the receptor for CXCL10, i.e. CXCR3, on MSCs was confirmed by qPCR and Western Blot. Moreover, CXCL10-guided MSC migration was assessed through an in vitro transwell migration assay. RESULTS: Intranasal MSC treatment at day 17 post-HI did not reduce lesion size in contrast to earlier treatment timepoints. Cerebral CXCL10 levels were significantly decreased at 17 days versus 10 days post-HI and correlated with reduced MSC migration towards the brain. In vitro experiments demonstrated that CXCR3 receptor inhibition prevented CXCL10-guided migration of MSCs. Intracranial CXCL10 injection at day 17 post-HI significantly increased the number of MSCs reaching the lesion which was accompanied by repair of the HI lesion as measured by reduced lesion size and glial scarring, and an increased number of neurons around the lesion. CONCLUSIONS: This study underscores the crucial role of the chemoattractant CXCL10 in guiding MSCs to the HI lesion after intranasal administration. Strategies to enhance CXCR3-mediated migration of MSCs may improve the efficacy of MSC therapy or extend its regenerative therapeutic window.

Bone Marrow Nucleated Cells and Bone Marrow-Derived CD271+ Mesenchymal Stem Cell in Treatment of Encephalopathy and Drug-Resistant Epilepsy.

The broad spectrum of brain injuries in preterm newborns and the plasticity of the central nervous system prompts us to seek solutions for neurodegeneration to prevent the consequences of prematurity and perinatal problems. The study aimed to evaluate the safety and efficacy of the implantation of autologous bone marrow nucleated cells and bone marrow mesenchymal stem cells in different schemes in patients with hypoxic-ischemic encephalopathy and immunological encephalopathy. Fourteen patients received single implantation of bone marrow nucleated cells administered intrathecally and intravenously, followed by multiple rounds of bone marrow mesenchymal stem cells implanted intrathecally, and five patients were treated only with repeated rounds of bone marrow mesenchymal stem cells. Seizure outcomes improved in most cases, including fewer seizures and status epilepticus and reduced doses of antiepileptic drugs compared to the period before treatment. The neuropsychological improvement was more frequent in patients with hypoxic-ischemic encephalopathy than in the immunological encephalopathy group. Changes in emotional functioning occurred with similar frequency in both groups of patients. In the hypoxic-ischemic encephalopathy group, motor improvement was observed in all patients and the majority in the immunological encephalopathy group. The treatment had manageable toxicity, mainly mild to moderate early-onset adverse events. The treatment was generally safe in the 4-year follow-up period, and the effects of the therapy were maintained after its termination.

Long-term recruitment of peripheral immune cells to brain scars after a neonatal insult.

Although brain scars in adults have been extensively studied, there is less data available regarding scar formation during the neonatal period, and the involvement of peripheral immune cells in this process remains unexplored in neonates. Using a murine model of neonatal hypoxic-ischemic encephalopathy (HIE) and confocal microscopy, we characterized the scarring process and examined the recruitment of peripheral immune cells to cortical and hippocampal scars for up to 1 year post-insult. Regional differences in scar formation were observed, including the presence of reticular fibrotic networks in the cortex and perivascular fibrosis in the hippocampus. We identified chemokines with chronically elevated levels in both regions and demonstrated, through a parabiosis-based strategy, the recruitment of lymphocytes, neutrophils, and monocyte-derived macrophages to the scars several weeks after the neonatal insult. After 1 year, however, neutrophils and lymphocytes were absent from the scars. Our data indicate that peripheral immune cells are transient components of HIE-induced brain scars, opening up new possibilities for late therapeutic interventions.

PKN1 Exerts Neurodegenerative Effects in an In Vitro Model of Cerebellar Hypoxic-Ischemic Encephalopathy via Inhibition of AKT/GSK3ß Signaling.

We recently identified protein kinase N1 (PKN1) as a negative gatekeeper of neuronal AKT protein kinase activity during postnatal cerebellar development. The developing cerebellum is specifically vulnerable to hypoxia-ischemia (HI), as it occurs during hypoxic-ischemic encephalopathy, a condition typically caused by oxygen deprivation during or shortly after birth. In that context, activation of the AKT cell survival pathway has emerged as a promising new target for neuroprotective interventions. Here, we investigated the role of PKN1 in an in vitro model of HI, using postnatal cerebellar granule cells (Cgc) derived from Pkn1 wildtype and Pkn1-/- mice. Pkn1-/- Cgc showed significantly higher AKT phosphorylation, resulting in reduced caspase-3 activation and improved survival after HI. Pkn1-/- Cgc also showed enhanced axonal outgrowth on growth-inhibitory glial scar substrates, further pointing towards a protective phenotype of Pkn1 knockout after HI. The specific PKN1 phosphorylation site S374 was functionally relevant for the enhanced axonal outgrowth and AKT interaction. Additionally, PKN1pS374 shows a steep decrease during cerebellar development. In summary, we demonstrate the pathological relevance of the PKN1-AKT interaction in an in vitro HI model and establish the relevant PKN1 phosphorylation sites, contributing important information towards the development of specific PKN1 inhibitors.

Oxidative Stress Markers in Human Brain and Placenta May Reveal the Timing of Hypoxic-Ischemic Injury: Evidence from an Immunohistochemical Study.

During pregnancy, reactive oxygen species (ROS) serve as crucial signaling molecules for fetoplacental circulatory physiology. Oxidative stress is thought to sustain the pathogenesis and progression of hypoxic-ischemic encephalopathy (HIE). A retrospective study was performed on the brains and placentas of fetuses and newborns between 36-42 weeks of gestation (Group_1: Fetal intrauterine deaths, Group_2: Intrapartum deaths, Group_3: Post-partum deaths, Control group sudden neonatal death); all groups were further divided into two subgroups (Subgroup_B [brain] and Subgroup_P [placenta]), and the study was conducted through the immunohistochemical investigations of markers of oxidative stress (NOX2, 8-OHdG, NT, iNOS), IL-6, and only on the brain samples, AQP4. The results for the brain samples suggest that NOX2, 8-OHdG, NT, iNOS, and IL-6 were statistically significantly expressed above the controls. iNOS was more expressed in the fetal intrauterine death (Group_1) and less expressed in post-partum death (Group_3), while in intrapartum death (Group_2), the immunoreactivity was very low. IL-6 showed the highest expression in the brain cortex of the fetal intrauterine death (Group_1), while intrapartum death (Group_2) and post-partum death (Group_3) showed weak immunoreactivity. Post-partum death (Group_3) placentas showed the highest immunoreactivity to NOX2, which was almost double that of the fetal intrauterine death (Group_1) and intrapartum death (Group_2) placentas. Placental tissues of fetal intrauterine death (Group_1) and intrapartum death (Group_2) showed higher expression of iNOS than post-partum death (Group_3), while the IL-6 expression was higher in the fetal intrauterine death (Group_1) than the post-partum death (Group_3). The AQP4 was discarded as a possible marker because the immunohistochemical reaction in the three groups of cases and the control group was negative. The goal of this study, from the point of view of forensic pathology, is to provide scientific evidence in cases of medical liability in the Obstetric field to support the clinical data of the timing of HIE.

Effect of Leuprolide Acetate, a GnRH Agonist, on Neuroinflammation and Anxiety-Like Behavior after Mild Hypoxic-Ischemic Encephalopathy in Rat Model.

BACKGROUND: Mild hypoxic-ischemic encephalopathy (HIE) is a condition that predisposes to negative outcomes such as neuroanatomical injury, mood disorders, and motor or cognitive disabilities. The neuroinflammation plays an important role in the neurological damage; therefore, reducing it could provide neuroprotection. The leuprolide acetate (LA) has shown to have neuroregenerative and immunomodulator properties in other nervous system injuries. OBJECTIVE: The aim of this study was to evaluate the immunomodulatory effect of LA in the acute phase of mild HIE and its effects in motor activity and behavior in a subacute phase. METHOD: Forty-five Wistar rats on postnatal day 7 were divided into Sham, HIE treated with saline solution (HIE-SS), and HIE-LA. The HIE was performed cutting of the right carotid artery followed by 60 min of hypoxia. The expression of the inflammatory cytokines interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and the chemokine CXCL-1 were evaluated 72 h after HIE by RT-qPCR and the motor activity and behavior were evaluated by open field test at postnatal day 33. RESULTS: HIE-SS animals showed increased expression of IL-1ß, TNF-α, IFN-γ, and CXCL-1 genes in injured tissue. However, the HIE-LA group exhibited similar expression levels of IL-1ß and TNF-α to the Sham group, while IFN-γ and CXCL-1 mRNA expression were attenuated with LA treatment. LA treatment also prevented anxiety-like behavior in the open field test. CONCLUSION: Treatment with LA partially reverses HIE-induced neuroinflammation and prevents anxiety-like behavior in neonatal rats.

Depressive-like behavior and impaired synaptic plasticity in the prefrontal cortex as later consequences of prenatal hypoxic-ischemic insult in rats.

Perinatal hypoxia-ischemia (HI) is a leading cause of morbidity and mortality among newborns. Infants with HI encephalopathy may experience lasting consequences, such as depression, in adulthood. In this study, we examined depressive-like behavior, neuronal population, and markers of monoaminergic and synaptic plasticity in the prefrontal cortex (PFC) of adolescent rats subjected to a prenatal HI model. Pregnant rats underwent a surgery in which uterine and ovarian blood flow was blocked for 45 min at E18 (HI procedure). Sham-operated subjects were also generated (SH procedure). Behavioral tests were conducted on male and female pups from P41 to P43, and animals were histologically processed or dissected for western blotting at P45. We found that the HI groups consumed less sucrose in the sucrose preference test and remained immobile for longer periods in the forced swim test. Additionally, we observed a significant reduction in neuronal density and PSD95 levels in the HI group, as well as a smaller number of synaptophysin-positive cells. Our results underscore the importance of this model in investigating the effects of HI-induced injuries, as it reproduces an increase in depressive-like behavior and suggests that the HI insult affects circuits involved in mood modulation.

[Artesunate alleviates hypoxic-ischemic brain damage in neonatal rats by inhibiting NLRP3 inflammasome activation and inflammatory cytokine secretion].

Objective To investigate the protective effect of artesunate on hypoxic-ischemic brain damage (HIBD) and its mechanism in neonatal rats. Methods 7-day-old neonatal SD rats were randomly divided into sham operation group, model group, artesunate 5 mg/kg group, artesunate 10 mg/kg group, artesunate 20 mg/kg group and dexamethasone 6 mg/kg group, with 18 rats in each group. HIBD models were established in groups except for the sham operation group. The sham operation group only needed to separate the left common carotid artery without ligation and nitrogen-oxygen mixed gas ventilation. Each group was injected with drug intraperitoneally right after surgery and the rats in the sham operation group and the model group were injected with an equal volume of normal saline (once a day for a total of 5 times). One hour after the last injection, the rats in each group were scored for neurological defects. After the rats were sacrificed, the brain water content was measured and the pathological changes of the brain tissues of rats were observed. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) was used to detect the neuronal cell apoptosis, and ELISA was applied to detect the levels of IL-1ß, IL-6 and TNF-α in brain tissues and peripheral blood of each group of rats. Western blot analysis was adopted to detect the protein expression levels of NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC) and caspase-1 in the rats brain tissues of each group. Results Compared with the model group, the neurological deficit score was decreased; the pathological damage of brain tissues was relieved; the brain water content was significantly reduced; the apoptosis number of hippocampal neurons was decreased significantly; the levels of IL-1ß, IL-6 and TNF-α in brain tissues and peripheral blood were significantly reduced; the protein expression levels of NLRP3, ASC and caspase-1 were significantly lowered in the middle-dose and high-dose artesunate groups and the dexamethasone group. Conclusion Artesunate can improve the neurological function, relieve the brain damage, and alleviate the brain edema in neonatal rats with HIBD. It can protect the HIBD, which may be related to the inhibition of NLRP3 inflammasome activation and reduction of inflammatory cytokine secretion.

Early evolution of glial morphology and inflammatory cytokines following hypoxic-ischemic injury in the newborn piglet brain.

Neuroinflammation is a hallmark of hypoxic-ischemic injury and can be characterized by the activation of glial cells and the expression of inflammatory cytokines and chemokines. Interleukin (IL)-1ß and tumor necrosis factor (TNF)α are among the best-characterized early response cytokines and are often expressed concurrently. Several types of central nervous system cells secrete IL-1ß and TNFα, including microglia, astrocytes, and neurons, and these cytokines convey potent pro-inflammatory actions. Chemokines also play a central role in neuroinflammation by controlling inflammatory cell trafficking. Our aim was to characterise the evolution of early neuroinflammation in the neonatal piglet model of hypoxic-ischemic encephalopathy (HIE). Piglets (< 24 h old) were exposed to HI insult, and recovered to 2, 4, 8, 12 or 24H post-insult. Brain tissue from the frontal cortex and basal ganglia was harvested for assessment of glial cell activation profiles and transcription levels of inflammatory markers in HI piglets with comparison to a control group of newborn piglets. Fluorescence microscopy was used to observe microglia, astrocytes, neurons, degenerating neurons and possibly apoptotic cells, and quantitative polymerase chain reaction was used to measure gene expression of several cytokines and chemokines. HI injury was associated with microglial activation and morphological changes to astrocytes at all time points examined. Gene expression analyses of inflammation-related markers revealed significantly higher expression of pro-inflammatory cytokines tumor necrosis factor-α (TNFα) and interleukin 1 beta (IL-1ß), chemokines cxc-chemokine motif ligand (CXCL)8 and CXCL10, and anti-inflammatory cytokine transforming growth factor (TGF)ß in every HI group, with some region-specific differences noted. No significant difference was observed in the level of C-X-C chemokine receptor (CCR)5 over time. This high degree of neuroinflammation was associated with a reduction in the number of neurons in piglets at 12H and 24H in the frontal cortex, and the putamen at 12H. This reduction of neurons was not associated with increased numbers of degenerating neurons or potentially apoptotic cells. HI injury triggered a robust early neuroinflammatory response associated with a reduction in neurons in cortical and subcortical regions in our piglet model of HIE. This neuroinflammatory response may be targeted using novel therapeutics to reduce neuropathology in our piglet model of neonatal HIE.

Impact of hydrogen gas inhalation during therapeutic hypothermia on cerebral hemodynamics and oxygenation in the asphyxiated piglet.

We previously reported the neuroprotective potential of combined hydrogen (H2) gas ventilation therapy and therapeutic hypothermia (TH) by assessing the short-term neurological outcomes and histological findings of 5-day neonatal hypoxic-ischemic (HI) encephalopathy piglets. However, the effects of H2 gas on cerebral circulation and oxygen metabolism and on prognosis were unknown. Here, we used near-infrared time-resolved spectroscopy to compare combined H2 gas ventilation and TH with TH alone. Piglets were divided into three groups: HI insult with normothermia (NT, n = 10), HI insult with hypothermia (TH, 33.5 ± 0.5 °C, n = 8), and HI insult with hypothermia plus H2 ventilation (TH + H2, 2.1-2.7%, n = 8). H2 ventilation and TH were administered and the cerebral blood volume (CBV) and cerebral hemoglobin oxygen saturation (ScO2) were recorded for 24 h after the insult. CBV was significantly higher at 24 h after the insult in the TH + H2 group than in the other groups. ScO2 was significantly lower throughout the 24 h after the insult in the TH + H2 group than in the NT group. In conclusion, combined H2 gas ventilation and TH increased CBV and decreased ScO2, which may reflect elevated cerebral blood flow to meet greater oxygen demand for the surviving neurons, compared with TH alone.

Effect of Hyperbaric oxygen on myelin injury and repair after hypoxic-ischemic brain damage in adult rat.

INTRODUCTION: Hypoxic-ischemic encephalopathy (HIE) is one of the leading causes of death and neurological disability with limited options for treatment in neonates, children and adults worldwide. The pathogenesis and treatment of white matter (WM) injury in adult patients with HIE remains largely elusive. METHODS: Sixty male Sprague-Dawley rats were randomly divided into control group, sham-operated group (HBO treatment 6 days after sham operation), and Hypoxia-ischemia (HI) induced brain damage group (receiving left carotid arteries ligation + hypoxia treatment), 1.5ATA hyperbaric oxygen group (HI + 1.5ATA HBOT) and 2.5ATA HBOT group (HI + 2.5ATA HBOT). All the rats were evaluated by water maze before operation, and 6 days after operation, and the function of learning and memory was evaluated; Demyelination in the hippocampus and prefrontal cortex was observed by Luxol fast blue staining (LFB) and MBP immunostaining; the number of Myelin Oligodendrocyte Glycoprotein (MOG),glial fibrillary acidic protein (GFAP), ionic calcium-binding adaptor (Iba-1) and NG2 positive cells in the hippocampus and prefrontal cortex were determined by immunofluorescence staining. The expression of interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor (TNF-α), Hypoxia Inducible Factor 1 Subunit Alpha (HIF1-α) and Superoxide dismutase (SOD) in brain and serum of rats were measured by Western Blot method and Enzyme linked immunosorbent assay (ELISA). RESULTS: Compared with those in the normal control group and sham-operated group, in the HI group, the learning and memory abilities of rats were significantly decreased (P < 0.05), the intensity of LFB and MBP immunostaining in hippocampus and prefrontal cortex was significantly decreased (P < 0.05); the number of MOG positive oligodendrocytes (OLs) significantly decreased (P < 0.05), whereas the number of Iba-1, GFAP, NG2 positive microglias, astrocytes and oligodendrocyte precursors (OPCs) was increased (P < 0.05); the level of IL-1ß, IL-6, TNF-α and HIF-1a in brain and serum were significantly increased (P < 0.05), whereas SOD was significantly decreased in brain and increased in serum. Compared with those in the HI group, in both 1.5ATA and 2.5ATA HBOT group, the learning and memory abilities were significantly increased (P < 0.05); the intensity of LFB and MBP immunostaining in the hippocampus and prefrontal cortex was significantly increased (P < 0.05); the number of MOG positive OLs significantly increased (P < 0.05); the number of Iba-1, GFAP, NG2 positive microglias, astrocytes and OPCs was decreased (P < 0.05); the level of IL-1ß, IL-6, TNF-α and HIF-1a in brain and serum were significantly decreased (P < 0.05); the level of SOD was significantly increased in brain and decreased in serum. Morever, compared with those in the 1.5ATA group, 2.5ATA provided better treatment results (P < 0.05). CONCLUSION: In the present study, we demonstrated the mechanism of different pressure HBOT on HI induced brain injury from three levels: (1) On a tissue level, HBOT protects against HI induced myelin injury; (2) On a cellular level, HBOT attenuates HI-induced OL loss, suppresss the reactive activation of astrocyte and microglia, and may promote OPC to differentiate into OL; (3) On a molecular level, HBOT inhibites neuroinflammation, and balances oxidative damage and antioxidant capacity. Among the above effects, 2.5ATA HBOT is better than 1.5ATA HBOT. Ongoing research will continue to seek out the signalling pathways and molecules mechanisms on different pressure of HBOT-related myelin protection, and possibly expand suitable HBOT use in adult HIE clinically.

Progesterone as a Neuroprotective Agent in Neonatal Hypoxic-Ischaemic Encephalopathy: A Systematic Review.

Hypoxic-ischaemic encephalopathy (HIE) in the newborn baby is a major contributor to neonatal mortality and morbidity across the world. Therapeutic hypothermia (TH) is the current standard treatment for moderate to severe HIE, but not all babies benefit. Potential neuroprotective actions of progesterone (PROG) include anti-apoptotic, anti-inflammatory, and anti-oxidative effects and reduction of energy depletion, tissue/cellular oedema, and excitotoxicity. In pre-clinical studies of neonatal HIE, PROG has neuroprotective properties but has not been the subject of systematic review. Here, our objective was to evaluate the evidence base for PROG as a potential therapeutic agent in HIE. The PICO framework was used to define the following inclusion criteria. Population: human neonates with HIE/animal models of HIE; intervention: PROG +/- other agents; comparison: V.S. control; outcome: pathological, neurobehavioural, and mechanistic outcome measures. Medline, EMBASE, and CINHAL were then searched between August to October 2018 using pre-defined medical subject heading and keywords. Study inclusion, data extraction, and risk of bias (ROB) analysis using the SYRCLE ROB tool were carried out by two authors. 14 studies were included in the review. They typically displayed a high ROB. This systematic review suggests that PROG reduced neuropathology and reduced neurobehavioural deficits post-hypoxic-ischaemic (HI) insult in 8 and 3 studies, respectively. However, there was sex dimorphism in the effects of PROG. In addition, there are limitations and biases in these studies, and there remains a need for well-designed large pre-clinical studies with greater methodological quality to further inform the efficacy, safety, dose, timing, and frequency of PROG administration. With such data, large animal studies could be planned combining PROG administration with and without TH.

Tumour necrosis factor blockade after asphyxia in foetal sheep ameliorates cystic white matter injury.

Cystic white matter injury is highly associated with severe neurodevelopmental disability and cerebral palsy in preterm infants, yet its pathogenesis remains poorly understood and there is no established treatment. In the present study, we tested the hypothesis that slowly evolving cystic white matter injury after hypoxia-ischaemia is mediated by programmed necrosis initiated by tumour necrosis factor. Tumour necrosis factor blockade was begun 3 days after hypoxia-ischaemia to target the tertiary phase of injury, when most secondary cell death is thought to be complete. Chronically instrumented preterm foetal sheep (0.7 gestation) received 25 min of hypoxia-ischaemia induced by complete umbilical cord occlusion or sham-umbilical cord occlusion (controls, n = 10), followed by intracerebroventricular infusion of the soluble TNF inhibitor, Etanercept, at 3, 8 and 13 days after umbilical cord occlusion (n = 9) or vehicle (n = 9). Foetal brains were processed for histology at 21 days after umbilical cord occlusion. Umbilical cord occlusion with vehicle was associated with a spectrum of macroscopic white matter degeneration, including white matter atrophy, ventriculomegaly and overt temporal lobe cystic white matter injury. Oligodendrocyte maturational arrest and impaired labelling of myelin proteins, characteristic of diffuse white matter injury, was observed in the parietal lobe and surrounding the cystic lesions in the temporal lobe. Etanercept markedly attenuated cystic white matter injury on the side of the intracerebroventricular infusion, with partial contralateral protection. Further, Etanercept improved oligodendrocyte maturation and labelling of myelin proteins in the temporal and parietal lobes. The present study shows that cystic white matter injury reflects late-onset tertiary cell death mediated by delayed neuroinflammation through the tumour necrosis factor pathway. Delayed tumour necrosis factor blockade markedly attenuated cystic white matter injury and restored oligodendrocyte maturation and deficits in myelin protein expression. These data suggest that delayed tumour necrosis factor blockade may represent a viable therapeutic strategy to reduce the risk of cystic and diffuse white matter injury and potentially cerebral palsy after preterm birth, with a surprisingly wide therapeutic window.

Therapeutic hypothermia demonstrates sex-dependent improvements in motor function in a rat model of neonatal hypoxic ischemic encephalopathy.

Neonatal hypoxic ischemic encephalopathy (HIE) is a neurological disease caused by restricted oxygen and blood flow to the brain at or around the time of birth. Long term cognitive and motor sequelae are common and demonstrate sexual dimorphism in animal studies. Therapeutic hypothermia (TH) is the standard of care for HIE, but provides incomplete neuroprotection. Using the Vannucci model of neonatal HIE, term-equivalent 11-day old rat pups were subjected to mild-moderate hypoxic-ischemic injury (HII), and a subset of animals were treated with TH. Sex-dependent neuroprotection was measured with gross and fine motor control assays, and functional deficits detected with these assays were correlated to injury in specific brain structures. At the equivalent of human adolescence and adulthood (P51-89), accelerod and beam walking tests were used to assess gross motor function, and string-pulling and food handling tests were used to assess fine motor function. At necropsy (P94-97), brain lesions were primarily focused to the posterior cerebrum and characterized by variable reduction in cortical, thalamic and hippocampal regions and glial scarring. Gross motor impairment was detected in male rats with untreated and TH-treated HIE in the accelerod test, but beam walk test data was confounded by the lower body mass of untreated male rats. HIE animals of both sexes demonstrated deficit in the forelimb contralateral to ischemic surgery, observed as unilaterally impaired food handling behaviors, and in string pulling as decreased string contacts and increased in bracing behavior. However, kinematic analyses revealed sex-specific decreases in peak speeds in string reaching and pulling movements. In both sexes, treatment with TH improved body mass, some measures of contralateral forelimb impairment, and the severity of brain lesions to levels not different to Sham surgery rats. Unique differences in behavior following TH were observed in female rats, who took longer to consume food items but traversed beams and approached strings faster than untreated and Sham females. Future use of these motor assays may unravel the subtle, sex-specific differences in HIE outcomes and in developing a customized therapeutic approach to neonatal brain injury.

Microglial iron trafficking: new player in brain injury.

BACKGROUND: Neonatal brain injury is a significant reason of neurodevelopmental abnormalities and long-term neurological impairments. Hypoxic-ischemic encephalopathy and preterm brain injury, including intraventricular hemorrhage are the most common grounds of brain injury for full-term and preterm neonates. The prevalence of hypoxic ischemic encephalopathy varies globally, ranging from 1 to 3.5/1000 live births in high-resource countries and 26/1000 in low-resource countries. Preterm birth's global incidence is 15 million, a significant reason for infant mortality and morbidity, permanent neurologic problems, and the associated social and economic burden. The widespread neurodevelopmental effects of neonatal brain injury could have an unfavorable impact on a variety of aspects of cognitive, linguistic, behavioral, sensory, and motor functions. Brain injury occurs via various mechanisms, including energy deprivation, excitatory amino acids, mitochondrial dysfunction, reactive oxygen species, and inflammation giving rise to different forms of cell death. The contribution of microglial activity in neonatal brain injury has widely been underlined by focusing on cell death mechanisms since the neuronal death pathways during their development are distinct from those in the adult brain. Iron accumulation and lipid peroxidation cause a relatively novel type of regulated cell death called ferroptosis. Neonates generally have biochemical iron inequalities, and their antioxidant potential is highly restricted, implying that ferroptosis may be significant in pathologic conditions. Moreover, inhaled nitric oxide therapy in infants may lead to microglial inflammation via ferroptosis and neuronal injury in the developing brain. This review article aims to summarize the studies that investigated the association between neonatal brain injury and iron metabolism, with a particular emphasis on the microglial activity and its application to the inhibition of neonatal brain injury.

Lack of Neuroprotection with a Single Intravenous Infusion of Human Amnion Epithelial Cells after Severe Hypoxia-Ischemia in Near-Term Fetal Sheep.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) around the time of birth results from loss of oxygen (hypoxia) and blood supply (ischemia). Exogenous infusion of multi-potential cells, including human amnion epithelial cells (hAECs), can reduce hypoxic-ischemic (HI) brain injury. However, there are few data on treatment of severe HI in large animal paradigms at term. The aim of the current study was to determine whether infusion of hAECs early after injury may reduce brain damage after ischemia in near-term fetal sheep. METHODS: Chronically instrumented fetal sheep (0.85 gestation) received 30 min of global cerebral ischemia followed by intravenous infusion of hAECs from 2 h after the end of ischemia (ischemia-hAEC, n = 6) or saline (ischemia-vehicle, n = 7). Sham control animals received sham ischemia with vehicle infusion (sham control, n = 8). RESULTS: Ischemia was associated with significant suppression of EEG power and spectral edge frequency until the end of the experiment and a secondary rise in cortical impedance from 24 to 72 h, which were not attenuated by hAEC administration. Ischemia was associated with loss of neurons in the cortex, thalamus, striatum and hippocampus, loss of white matter oligodendrocytes and increased microglial numbers in the white matter, which were not affected by hAEC infusion. CONCLUSIONS: A single intravenous administration of hAECs did not reduce electrographic or histological brain damage after 30 min of global cerebral ischemia in near-term fetal sheep.

No Differences in Cerebral Immunohistochemical Markers following Remote Ischemic Postconditioning in Newborn Piglets with Hypoxia-Ischemia.

BACKGROUND: Despite therapeutic hypothermia, neonates with hypoxic-ischemic encephalopathy still develop neurological disabilities. We have previously investigated neuroprotection by remote ischemic postconditioning (RIPC) in newborn piglets following hypoxia-ischemia (HI). The aim of this study was to further investigate potential effects of RIPC on cerebral immunohistochemical markers related to edema, apoptosis, and angiogenesis. METHODS: Brain expression of aquaporin 4, caspase-3, B-cell lymphoma 2, and vascular endothelial growth factor was analyzed by immunohistochemistry in 23 piglets, randomly selected from a larger study of RIPC after HI. Twenty animals were subjected to 45 minutes of HI and randomized to treatment with and without RIPC, while three animals were randomized to sham procedures. RIPC was conducted by four conditioning cycles of 5-minute ischemia and reperfusion. Piglets were euthanized 72 hours after the HI insult. RESULTS: Piglets subjected to HI treated with and without RIPC were similar at baseline and following the HI insult. However, piglets randomized to HI alone had longer duration of low blood pressure during the insult. We found no differences in the brain expression of the immunohistochemical markers in any regions of interest or the whole brain between the two HI groups. CONCLUSION: RIPC did not influence brain expression of markers related to edema, apoptosis, or angiogenesis in newborn piglets at 72 hours after HI. These results support previous findings of limited neuroprotective effect by this RIPC protocol. Our results may have been affected by the time of assessment, use of fentanyl as anesthetic, or limitations related to our immunohistochemical methods.

The prolyl hydroxylase inhibitor GSK1120360A reduces early brain injury, but protection is not maintained in a neonatal rat model of hypoxic ischaemic encephalopathy.

Hypoxic-ischemic encephalopathy (HIE) in newborns is associated with high morbidity and mortality, with many babies suffering long-term neurological deficits. Currently, treatment options are limited to therapeutic hypothermia, which is not appropriate for use in all babies. Previous studies have shown protective effects of increasing the transcription factor-hypoxia-inducible factor-1 (HIF-1) in animal models, by using mild hypoxia or compounds that act as prolyl hydroxylase inhibitors (PHIs). Here, we aimed to examine the neuroprotective actions of an orally active, small molecule PHI, GSK1120360A in a neonatal rat model of hypoxia-ischemia (HI) compared to another PHI, desferrioxamine (DFX). Sprague-Dawley rats underwent HI surgery on postnatal day 7 (P7), where unilateral carotid artery occlusion was performed followed by hypoxia (8% oxygen, 3 h). Initial testing showed that GSK1120360A and erythropoietin levels were detectable in plasma at 6 h following oral exposure to GSK1120360A. For the short-term neuroprotection study, pups were assigned to receive either saline (s.c), desferrioxamine (DFX-200 mg/kg, s.c), methylcellulose (1%, oral) or GSK1120360A (30 mg/kg, oral) immediately after HI. Histological analysis showed that GSK1120360A in this setting reduced brain injury size 7 days after HI, compared to the methylcellulose vehicle control group. DFX had no significant effect on injury size compared to saline group at the same 7 day timepoint. In the long-term neuroprotection study, pups were randomly assigned to be administered methylcellulose (1%, oral) or GSK1120360A (30 mg/kg, oral) immediately after HI. On P42, rats underwent behavioural testing using the forelimb grip strength, grid walking and novel object recognition tasks, and brains were collected for histological analysis. Long-term behavioural deficits were observed in grid walking, grip strength and novel object recognition tests after HI which were not improved in the GSK1120360A treatment group compared to the methylcellulose group. Similarly, there was no improvement in injury size on P42 in the GSK1120360A study group compared to the methylcellulose group. Here, we have shown that GSK1120360A can reduce brain injury at 7 days but that this neuroprotective benefit is not maintained when examined at 5 weeks after HI.

Novel Injury Scoring Tool for Assessing Brain Injury following Neonatal Hypoxia-Ischemia in Mice.

The variability of severity in hypoxia-ischemia (HI)-induced brain injury among research subjects is a major challenge in developmental brain injury research. Our laboratory developed a novel injury scoring tool based on our gross pathological observations during hippocampal extraction. The hippocampi received scores of 0-6 with 0 being no injury and 6 being severe injury post-HI. The hippocampi exposed to sham surgery were grouped as having no injury. We have validated the injury scoring tool with T2-weighted MRI analysis of percent hippocampal/hemispheric tissue loss and cell survival/death markers after exposing the neonatal mice to Vannucci's rodent model of neonatal HI. In addition, we have isolated hippocampal nuclei and quantified the percent good quality nuclei to provide an example of utilization of our novel injury scoring tool. Our novel injury scores correlated significantly with percent hippocampal and hemispheric tissue loss, cell survival/death markers, and percent good quality nuclei. Caspase-3 and Poly (ADP-ribose) polymerase-1 (PARP1) have been implicated in different cell death pathways in response to neonatal HI. Another gene, sirtuin1 (SIRT1), has been demonstrated to have neuroprotective and anti-apoptotic properties. To assess the correlation between the severity of injury and genes involved in cell survival/death, we analyzed caspase-3, PARP1, and SIRT1 mRNA expressions in hippocampi 3 days post-HI and sham surgery, using quantitative reverse transcription polymerase chain reaction. The ipsilateral (IL) hippocampal caspase-3 and SIRT1 mRNA expressions post-HI were significantly higher than sham IL hippocampi and positively correlated with the novel injury scores in both males and females. We detected a statistically significant sex difference in IL hippocampal caspase-3 mRNA expression with comparable injury scores between males and females with higher expression in females.

Cerebral perfusion changes of the basal ganglia and thalami in full-term neonates with hypoxic-ischaemic encephalopathy: a three-dimensional pseudo continuous arterial spin labelling perfusion magnetic resonance imaging study.

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the common causes of neurological injury in full-term neonates following perinatal asphyxia. The conventional magnetic resonance technique has low sensitivity in detecting variations in cerebral blood flow in patients with HIE. OBJECTIVE: This article evaluates the clinical diagnostic value of three-dimensional pseudo-continuous arterial spin labelling (3-D pcASL) perfusion magnetic resonance imaging (MRI) for early prediction of neurobehavioral outcomes in full-term neonates with HIE. MATERIALS AND METHODS: All neonates diagnosed with HIE underwent MRI (conventional and 3-D pcASL perfusion MRI). Cerebral blood flow values were measured in the basal ganglia (caudate nuclei, lenticular nuclei), thalami and white matter regions (frontal lobes, corona radiata). After 1-month follow-up, the Neonatal Behavioral Neurological Assessment scores were used to divide patients into favourable outcome group versus adverse outcome group. RESULTS: Twenty-three patients were enrolled in this study. There were no statistical differences between the symmetrical cerebral blood flow values of bilateral basal ganglia, thalami and white matter regions. However, the cerebral blood flow values of grey matter nuclei were higher than the white matter regions. The average value of cerebral blood flow in the basal ganglia and thalami in the adverse outcome group was 37.28±6.42 ml/100 g/min, which is greater than the favourable outcome group (22.55 ± 3.21 ml/100 g/min) (P<0.01). The area under the curve (AUC) of 3-D pcASL perfusion MRI was 0.992 with a cutoff value of 28.75 ml/100 g/min, with a Youden's index of 0.9231. The sensitivity and specificity were 92.3% and 100%, respectively. CONCLUSION: The 3-D pcASL demonstrated higher perfusion alteration in the basal ganglia and thalami of neonatal HIE with adverse outcomes. The 3-D pcASL perfusion MRI has the potential to predict neurobehavioral outcomes of neonates with HIE.