Neonatal Hypoxic-ischemic Encephalopathy Model Research Articles

Structural and Functional Effects of C5aR1 Antagonism in a Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy

Plain Language SummaryIn this study, we tested a new treatment for a disease in babies called hypoxic-ischemic encephalopathy (HIE). HIE is caused by restricted oxygen and blood flow around the time of birth, and most damage is caused when blood flow is restored. One reason for this is excess inflammation, which happens because a large amount of specific proteins and chemicals are released. Some inflammation is protective, but excessive inflammation injures healthy tissue, and in HIE this contributes to lifelong physical and mental disabilities, even death. The only treatment for HIE is cooling therapy, but even with this treatment many infants still do not survive or live with permanent disability. We mimicked the disease in rats and then gave them a drug that prevents excess inflammation by binding and disabling specific proteins. We measured improvement by comparing physical and mental abilities and brain damage in the treated rats to untreated and normal rats. Overall, the drug improved many but not all skills and parts of the brain harmed by the disease. More testing needs to be done to determine the optimal dose and if there is additional benefit when the drug is combined with cooling therapy. In closing, our results show this treatment warrants further development and could help infants grow up with less hardship and lead better, healthier lives.

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.

Effect of Clemastine on Neurophysiological Outcomes in an Ovine Model of Neonatal Hypoxic-Ischemic Encephalopathy.

Originally approved by the U.S. Food and Drug Administration (FDA) for its antihistamine properties, clemastine can also promote white matter integrity and has shown promise in the treatment of demyelinating diseases such as multiple sclerosis. Here, we conducted an in-depth analysis of the feasibility, safety, and neuroprotective efficacy of clemastine administration in near-term lambs (n = 25, 141-143 days) following a global ischemic insult induced via an umbilical cord occlusion (UCO) model. Lambs were randomly assigned to receive clemastine or placebo postnatally, and outcomes were assessed over a six-day period. Clemastine administration was well tolerated. While treated lambs demonstrated improvements in inflammatory scores, their neurodevelopmental outcomes were unchanged.

Use of liposome-encapsulated estetrol for treatment of neonatal hypoxic-ischemic encephalopathy

Estetrol (E4) is a natural estrogen synthesized only during pregnancy. It has strong neuroprotective and antioxidative activities. The aim of the present study was to define the neuroprotective potency of E4 encapsulated either in liposome (Lipo-E4) or in drug-in cyclodextrin (HP-β-CD) in liposome (DCL) system, and compare them with a single use of E4. In vitro studies were performed in an oxidative stress model of primary hippocampal neuronal cell cultures, followed by the lactate dehydrogenase activity and cell proliferation assays. In vivo studies were conducted by using a model of neonatal hypoxic-ischemic encephalopathy in immature rat pups. Brain samples were studied by (immuno)histochemistry for the detection of survived cells, expression of microtubule-associated protein-2, myelin basic protein, doublecortin and vascular-endothelial growth factor. Concentrations of glial fibrillary acidic protein in blood serum were studied by ELISA. In vitro, cell proliferation was significantly up-regulated in cultures treated either by DCL-E4 or E4 compared to the control cells, whereas DCL-E4 treated cells had significantly higher survival rate than the cells treated by E4 alone. Evaluation of brain samples showed that DCL-E4 and a high dose of E4 alone significantly preserve the grey and the white matter loses, and diminish GFAP expression in blood. Although DCL-E4 and E4 have similar effect on neurogenesis in the hippocampus and the cortex, DCL-E4 treatment significantly up-regulates angiogenesis in the hippocampus compared to a single use of E4. Present work reveals for the first time that liposome-encapsulated E4 might be a better alternative to a single use of E4.

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)-1β 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-1β 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-1β), chemokines cxc-chemokine motif ligand (CXCL)8 and CXCL10, and anti-inflammatory cytokine transforming growth factor (TGF)β 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.

Transcriptional regulation of NRF1 on metabotropic glutamate receptors in a neonatal hypoxic‑ischemic encephalopathy rat model.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a kind of brain injury that causes severe neurological disorders in newborns. Metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs) are significantly associated with HIE and are involved in ischemia-induced excitotoxicity. This study aimed to investigate the upstream mechanisms of mGluRs and the transcriptional regulation by nuclear respiratory factor 1 (NRF1). The rat model of neonatal HIE was created using unilateral carotid artery ligation and in vitro oxygen-glucose deprivation paradigm. We used western blot, immunofluorescence, Nissl staining, and Morris water maze to investigate the impact of NRF1 on brain damage and learning memory deficit by HIE. We performed ChIP and luciferase activities to identify the transcriptional regulation of NRF1 on mGluRs. The neuronal NRF1 and some glutamatergic genes expression synchronously declined in infarcted tissues. The NRF1 overexpression effectively restored the expression of some glutamatergic genes and improved cognitive performance. NRF1 regulated some members of mGluRs and iGluRs in hypoxic-ischemic neurons. Finally, NRF1 is bound to the promoter regions of Grm1, Grm2, and Grm8 to activate their transcription. NRF1 is involved in the pathology of the neonatal HIE rat model, suggesting a novel therapeutic approach to neonatal HIE. NRF1 and some glutamatergic genes were synchronously downregulated in the infarcted brain of the neonatal HIE rat model. NRF1 overexpression could rescue cognitive impairment caused by the neonatal HIE rat model. NRF1 regulated the expressions of Grm1, Grm2, and Grm8, which activated their transcription by binding to the promoter regions.

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.

Changes of Oxidative Stress-Related Gene Expression in an in vitro Model of Neonatal Hypoxic-Ischemic Encephalopathy

Background: We recently demonstrated that oxygen-glucose deprivation (OGD) and unconjugated bilirubin (UCB) can damage mature and immature organotypic hippocampal slices and induce an oxidative stress similar to what occurs in jaundiced term and preterm infants with hypoxic-ischemic encephalopathy (HIE). Objectives: To assess the effects of OGD and UCB on the expression of heme-oxygenase 1 (HO-1) and oxidative stress-related enzymes in an in vitro model of HIE. Methods: Mature and immature organotypic hippocampal slices were exposed to 30-min OGD and to 24 h UCB or UCB plus human serum albumin (HSA). The expression of HO-1, superoxide dismutase 1 (SOD1), catalase (CAT), glutathione peroxidase (GPX), and nuclear factor erythroid-related factor 2 were analyzed by real-time PCR. Results: In mature slices, OGD did not affect the expression of HO-1 and oxidative stress-induced enzymes. The addition of UCB was associated with the upregulation of HO-1 and Nrf2 that is abolished by the presence of equimolar amount of HSA. In immature slices, OGD induced the downregulation of CAT, GPX, and Nrf2 expression and the addition of UCB further decreased GPX. The addition of UCB and HSA reverted the effects of OGD and UCB on gene expression. Conclusions: In an in vitro model of HIE in term infants, we did not observe neuroprotective changes of the expression of HO-1 and genes involved in antioxidant defenses. Conversely, in an in vitro model of HIE in preterm infants, we observed a harmful decrease of the expression of genes encoding for antioxidant enzymes.

The protective effect of MiR-27a on the neonatal hypoxic-ischemic encephalopathy by targeting FOXO1 in rats.

BackgroundNeonatal hypoxic-ischemic encephalopathy (HIE), a kind of hypoxic-ischemic brain damage caused by perinatal asphyxia, is the most crucial cause of neonatal death and long-term neurological dysfunction in children. We aimed to investigate the protective effects of micro (mi)R-27a on HIE in neonatal rats.MethodsA rat model of neonatal HIE was constructed by modification of the Rice-Vannucci model. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to test the expressions of miR-27a, FOXO1 messenger RNA (mRNA), interleukin-1β (IL-1β) mRNA, and tumor necrosis factor-α (TNF-α) mRNA, and western blot was applied to test the expression of FOXO1. In order to overexpress miR-27a, an intracerebroventricular injection (i.c.v) of miR-27a mimic was administered. We adopted 2,3,5-triphenytetrazolium chloride (TTC) staining and brain water content measurement to test the effects of miR-27a on the infarcted volume and edema in brain after HIE. Flow cytometry (FCM) analysis was applied to test the effects of miR-27a on the infiltrated peripheral immune cells in the rat brains after HIE.ResultsWe successfully established a rat model of neonatal HIE. It was revealed that the expressions of miR-27a decreased gradually after HIE, however, the expressions of FOXO1 mRNA increased. After injection of the miR-27a mimic, the expression of miR-27a in the rat HIE model brains was significantly upregulated, however, the expression of FOXO1 was robustly downregulated. Both TTC staining and brain water content showed that the infarcted volume and brain edema was markedly increased after HIE. Interestingly, the overexpression of miR-27a reduced the infarcted volume and edema induced by HIE. Additionally, RT-qPCR and FCM analysis showed that HIE lead to increases of IL-1β, TNF-α, and infiltrated immune cells. Overexpression of miR-27a could reduce the expressions of IL-1β mRNA and TNF-α mRNA, and the cell numbers of infiltrated peripheral macrophages and neutrophils in the brain.ConclusionsMiR-27a plays protective roles by reducing infarct volume and brain edema, and inhibiting inflammatory factors and infiltrated peripheral immune cells by targeting FOXO1 in neonatal HIE rats.

Combining Hypothermia and Oleuropein Subacutely Protects Subcortical White Matter in a Swine Model of Neonatal Hypoxic-Ischemic Encephalopathy.

Neonatal hypoxia-ischemia (HI) causes white matter injury that is not fully prevented by therapeutic hypothermia. Adjuvant treatments are needed. We compared myelination in different piglet white matter regions. We then tested whether oleuropein (OLE) improves neuroprotection in 2- to 4-day-old piglets randomized to undergo HI or sham procedure and OLE or vehicle administration beginning at 15 minutes. All groups received overnight hypothermia and rewarming. Injury in the subcortical white matter, corpus callosum, internal capsule, putamen, and motor cortex gray matter was assessed 1 day later. At baseline, piglets had greater subcortical myelination than in corpus callosum. Hypothermic HI piglets had scant injury in putamen and cerebral cortex. However, hypothermia alone did not prevent the loss of subcortical myelinating oligodendrocytes or the reduction in subcortical myelin density after HI. Combining OLE with hypothermia improved post-HI subcortical white matter protection by preserving myelinating oligodendrocytes, myelin density, and oligodendrocyte markers. Corpus callosum and internal capsule showed little HI injury after hypothermia, and OLE accordingly had minimal effect. OLE did not affect putamen or motor cortex neuron counts. Thus, OLE combined with hypothermia protected subcortical white matter after HI. As an adjuvant to hypothermia, OLE may subacutely improve regional white matter protection after HI.

Rh-CSF1 Attenuates Oxidative Stress and Neuronal Apoptosis via the CSF1R/PLCG2/PKA/UCP2 Signaling Pathway in a Rat Model of Neonatal HIE.

Oxidative stress (OS) and neuronal apoptosis are major pathological processes after hypoxic-ischemic encephalopathy (HIE). Colony stimulating factor 1 (CSF1), binding to CSF1 receptor (CSF1R), has been shown to reduce neuronal loss after hypoxic-ischemia- (HI-) induced brain injury. In the present study, we hypothesized that CSF1 could alleviate OS-induced neuronal degeneration and apoptosis through the CSF1R/PLCG2/PKA/UCP2 signaling pathway in a rat model of HI. A total of 127 ten-day old Sprague Dawley rat pups were used. HI was induced by right common carotid artery ligation with subsequent exposure to hypoxia for 2.5 h. Exogenous recombinant human CSF1 (rh-CSF1) was administered intranasally at 1 h and 24 h after HI. The CSF1R inhibitor, BLZ945, or phospholipase C-gamma 2 (PLCG2) inhibitor, U73122, was injected intraperitoneally at 1 h before HI induction. Brain infarct volume measurement, cliff avoidance test, righting reflex test, double immunofluorescence staining, western blot assessment, 8-OHdG and MitoSOX staining, Fluoro-Jade C staining, and TUNEL staining were used. Our results indicated that the expressions of endogenous CSF1, CSF1R, p-CSF1R, p-PLCG2, p-PKA, and uncoupling protein2 (UCP2) were increased after HI. CSF1 and CSF1R were expressed in neurons and astrocytes. Rh-CSF1 treatment significantly attenuated neurological deficits, infarct volume, OS, neuronal apoptosis, and degeneration at 48 h after HI. Moreover, activation of CSF1R by rh-CSF1 significantly increased the brain tissue expressions of p-PLCG2, p-PKA, UCP2, and Bcl2/Bax ratio, but reduced the expression of cleaved caspase-3. The neuroprotective effects of rh-CSF1 were abolished by BLZ945 or U73122. These results suggested that rh-CSF1 treatment attenuated OS-induced neuronal degeneration and apoptosis after HI, at least in part, through the CSF1R/PLCG2/PKA/UCP2 signaling pathway. Rh-CSF1 may serve as therapeutic strategy against brain damage in patients with HIE.

Temporally Altered miRNA Expression in a Piglet Model of Hypoxic Ischemic Brain Injury.

Hypoxic ischemic encephalopathy (HIE) is the most frequent cause of acquired infant brain injury. Early, clinically relevant biomarkers are required to allow timely application of therapeutic interventions. We previously reported early alterations in several microRNAs (miRNA) in umbilical cord blood at birth in infants with HIE. However, the exact timing of these alterations is unknown. Here, we report serial changes in six circulating, cross-species/bridging biomarkers in a clinically relevant porcine model of neonatal HIE with functional analysis. Six miRNAs—miR-374a, miR-181b, miR-181a, miR-151a, miR-148a and miR-128—were significantly and rapidly upregulated 1-h post-HI. Changes in miR-374a, miR-181b and miR-181a appeared specific to moderate-severe HI. Histopathological injury and five miRNAs displayed positive correlations and were predictive of MRS Lac/Cr ratios. Bioinformatic analysis identified that components of the bone morphogenic protein (BMP) family may be targets of miR-181a. Inhibition of miR-181a increased neurite length in both SH-SY5Y cells at 1 DIV (days in vitro) and in primary cultures of rat neuronal midbrain at 3 DIV. In agreement, inhibition of miR-181a increased expression of BMPR2 in differentiating SH-SY5Y cells. These miRNAs may therefore act as early biomarkers of HIE, thereby allowing for rapid diagnosis and timely therapeutic intervention and may regulate expression of signalling pathways vital to neuronal survival.Electronic supplementary materialThe online version of this article (10.1007/s12035-020-02018-w) contains supplementary material, which is available to authorized users.

MicroRNA-374a-5p inhibits neuroinflammation in neonatal hypoxic-ischemic encephalopathy via regulating NLRP3 inflammasome targeted Smad6

AimsNeonatal hypoxic-ischemic encephalopathy (HIE) is still an important cause of neurological dysfunction. At present, there is no reliable biochemical index in clinical examination. Increasing evidence demonstrates that microRNAs (miRNAs) are involved in the process of HIE, and miR-374a-5p is down-regulated in HIE infants. In this study, the aim is to investigate the role and mechanism of miR-374a-5p in HIE. Main methodsSprague-Dawley (SD) rats were used to establish model of neonatal HIE, pathologic changes and inflammatory response of brain tissues were measured. Subsequently, primary microglia were induced by LPS (1 μg/ml) in vitro, the miR-374a-5p mimic, Ad-Smad6 adenovirus vector and NLRP3 siRNA oligo were applied for microglial transfection. Furthermore, the target relationship between miR-374a-5p and Smad6 was analyzed, while microglia activity and inflammatory factor (IL-1β, TNF-α and IL-6) levels were detected. Key findingsHerein, we found that over-expression of miR-374a-5p significantly attenuated brain injury and strongly inhibited the release of pro-inflammatory cytokines in neonatal rat HIE model. In vitro, miR-374a-5p inhibited LPS-induced microglial pro-inflammatory cytokines production by regulating NLRP3 inflammasome. In addition, Smad6 was identified as a direct target of miR-374a-5p, and miR-374a-5p had a negative regulatory effect on Smad6 expression. By targeting Smad6, miR-374a-5p inhibited the activation of NLRP3 inflammatory signals in microglia and the subsequent release of pro-inflammatory factors. SignificanceOur study recognized that miR-374a-5p as a novel regulator of microglial activation in neonatal HIE highlighted potential therapeutic target for the treatment of neonatal hypoxic-ischemic brain injury.

Combined Treatment with Insulin-Like Growth Factor 1 and AMD3100 Improves Motor Outcome in a Murine Model of Neonatal Hypoxic-Ischemic Encephalopathy

Stem cell transplantation is a promising intervention for neonatal hypoxic-ischemic encephalopathy (HIE); however, universal feasibility and safety have not been thoroughly evaluated. AMD3100 and insulin-like growth factor 1 (IGF1) mobilize progenitor cells into peripheral circulation. The objective of this study was to assess the short-term efficacy of inducing endogenous stem cell mobilization after injury in a model of neonatal HIE. Postnatal day 9 CD1 pups received sham surgery or unilateral carotid artery ligation and 30 min of hypoxia followed by saline, AMD3100, IGF1, or both agents. Intraperitoneal injections of 5-ethynyl-2′-deoxy­uridine (EdU) and 5-bromo-2′-deoxyuridine were used to ­label replicating progenitor cells. At P14, animals underwent rotarod testing, and the brains were sectioned for area measurements and immunofluorescence staining. Comparisons were made using one-way analysis of variance. Spearman’s rho was calculated to assess correlation between rotarod results and markers of brain injury. Pups treated with both agents had improved rotarod performance (p = 0.02) and increased EdU+ progenitor cells in the subgranular zone (SGZ) compared to injured controls (p = 0.10). An increase in active cells within the SGZ was correlated with improved rotarod performance (r = 0.84, p = 0.04). There were no differences in overall injury score or in brain area or number of activated cells in the subventricular zone between the treatment groups. Combined treatment with AMD3100 and IGF1 shows promise for decreasing brain injury and improving motor function in pups after HIE which correlated with changes in the number of active progenitor cells in the SGZ.

A single-nucleotide polymorphism induced alternative splicing in Tacr3 involves in hypoxic-ischemic brain damage

Single-nucleotide polymorphism (SNP) and Alternative splicing (AS) were found to be implicated in certain diseases, nevertheless, the contributions of mRNA SNPs and AS to pathogenesis in developing rat brains with hypoxic-ischemic encephalopathy (HIE) remained largely vague. Additionally, the disease associated with Tacr3 was normosmic congenital hypogonadotropic hypogonadism, while the relationship between HIE and Tacr3 remained largely elusive. The current study was designed to investigate the differentially expressed mRNAs and related SNPs as well as AS in neonatal rats subjected to HIE to identify if the exhibition of AS was associated with SNPs under pathological condition. Firstly, we used postnatal day 7 Sprague-Dawley rats to construct neonatal HIE model, and analyzed the expression profiles of SNP mRNA in hypoxic-ischemic (HI) and sham brains by using RNA sequencing. Then four genes, including Mdfic, Lpp, Bag3 and Tacr3, connecting with HIE and exhibiting SNPs and AS were identified by bioinformatics analysis. Moreover, combined with exonic splicing enhancer (ESE) and alternative splice site predictor (ASSP) analysis, we found that Tacr3 is associated specifically with HIE through 258547789 G > A SNP in inside the Alt First Exon and 258548573 G > A SNP in outside the Alt First Exon. Taken together, our study provides new evidence to understand the role of Tacr3 in HIE and it is possibly a potential target for the treatment of HIE in future clinic trial.

Ghrelin attenuates oxidative stress and neuronal apoptosis via GHSR-1α/AMPK/Sirt1/PGC-1α/UCP2 pathway in a rat model of neonatal HIE

Neuronal apoptosis induced by oxidative stress is one of the major pathological processes involved in neurological impairment after hypoxic-ischemic encephalopathy (HIE). Ghrelin, the unique endogenous ligand for the growth hormone secretagogue receptor-1α (GHSR-1α), could take an anti-apoptotic role in the brain. However, whether ghrelin can attenuate neuronal apoptosis by attenuating oxidative stress after hypoxia-ischemia (HI) insult remains unknown. To investigate the beneficial effects of ghrelin on oxidative stress injury and neuronal apoptosis induced by HI, ten-day old unsexed rat pups were subjected to HI injury and exogenous recombinant human ghrelin(rh-Ghrelin) was administered intranasally at 1 h and 24 h after HI induction. [D-Lys3]-GHRP-6, a selective inhibitor of GHSR-1α and Ex527, a selective inhibitor of GHSR-1α were administered intranasally at 1 h before HI induction respectively. Small interfering ribonucleic acid (siRNA) for GHSR-1α were administered by intracerebroventricular (i.c.v) injection at 24 h before HI induction. Neurological tests, immunofluorescence, MitoSox staining, Fluoro-Jade C staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and western blot experiments were performed. Our results indicated that ghrelin significantly improved neurobehavioral outcomes and reduced oxidative stress and neuronal apoptosis. Moreover, ghrelin treatment significantly promoted phosphorylation of AMPK, upregulated the expression of Sirt1, PGC-1α, UCP2 and the ratio of Bcl2/Bax, while it downregulated cleaved caspase-3 levels. The protective effects of ghrelin were reversed by [D-Lys3]-GHRP-6, GHSR-1α siRNA or Ex527. In conclusion, our data demonstrated that ghrelin reduced oxidative stress injury and neuronal apoptosis which was in part via the GHSR-1α/AMPK/Sirt1/PGC-1α/UCP2 signalling pathway after HI. Ghrelin may be a novel therapeutic target for treatment after neonatasl HI injury.

Suppression of PDGF induces neuronal apoptosis after neonatal cerebral hypoxia and ischemia by inhibiting P-PI3K and P-AKT signaling pathways

Neonatal hypoxic-ischemic encephalopathy (HIE) always results in severe neurologic dysfunction, nevertheless effective treatments are limited and the underlying mechanism also remains unclear. In this study, we firstly established the neonatal HIE model in the postnatal day 7 SD rats, Zea-Longa score and TTC staining were employed to assess the neurological behavior and infarct volume of the brain after cerebral hypoxia-ischemia (HI). Afterwards, protein chip was adopted to detect the differential proteins in the right cortex, hippocampus and lung, ultimately, PDGF was noticed. Then, immunohistochemistry, immunofluorescence double staining of NeuN/PDGF, and western blot were used to validate the expression level of PDGF in the cortex and hippocampus at 6 hours (h), 12 h and 24 h after HI. To determine the role of PDGF, the primary cortical neurons were prepared and performed PDGF shRNA administration. The results showed that HIE induced a severe behavioral dysfunction and brain infarction in neonatal rats, and the expression of PDGF in right cortex and hippocampus was remarkably increased after HI. Whereas, suppressing PDGF resulted in a significant loss of neurons and inhibition of neurite growth. Moreover, the protein level of P-PI3K and P-AKT signaling pathways were largely decreased following PDGF-shRNA application in the cortical neurons. In conclusion, PDGF suppression aggravated neuronal dysfunction, and the underlying mechanism is associated with inhibiting the phosphorylation of P-PI3K and P-AKT. Together, PDGF regulating PI3K and AKT may be an important panel in HIE events and therefore may provide possible strategy for the treatment of HIE in future clinic trail.

Neonatal hypoxia-ischemia caused mild motor dysfunction, recovered by acrobatic training, without affecting morphological structures involved in motor control in rats

The aim of this study was to evaluated motor function and morphological aspects of the components involved in motor control (sensorimotor cortex, spinal cord, sciatic nerve, neuromuscular junctions and skeletal muscle) in male Wistar rats exposed to a model of neonatal hypoxic-ischemic encephalopathy (HIE) and the possible influence of different physical exercise protocols – treadmill and acrobatic. Male Wistar rats at the 7th post-natal day (PND) were submitted to the HIE model and from the 22nd until 60th PND the exercise protocols (treadmill or acrobatic training) were running. After the training, the animals were evaluated in Open Field, Ladder Rung Walking and Rotarod tasks and after samples of the motor control components were collected. Our results evidenced that the acrobatic training reversed the hyperactivity and anxiety, caused locomotion improvement and decreased brain atrophy in HIE animals. We did not find morphological differences on sensorimotor cortex, spinal cord, sciatic nerve, neuromuscular junctions and skeletal muscle in the animals submitted to HIE model. These intriguing data support the statement of the Rice-Vannucci model does not seem to reproduce, in structures involved in control function, the damage found in humans that suffer HIE. Regarding the protocols of exercise, we proposed that the acrobatic exercise could be a good therapeutic option especially in children affected by neonatal HIE and can be responsible for good results in cognitive and motor aspects.

Evaluating a Targeted Bedside Measure of Cerebral Perfusion in a Nonhuman Primate Model of Neonatal Hypoxic‐Ischemic Encephalopathy

To compare ultrasound-derived resistive indices (RIs) obtained at the level of the thalamus via fast Doppler ultrasound with traditional anterior cerebral artery measures in a model of neonatal hypoxic-ischemic encephalopathy and to correlate each with clinical outcomes. Nine nonhuman primate neonates underwent no umbilical cord occlusion (n = 3), umbilical cord occlusion without hypothermia (n = 3), or umbilical cord occlusion with hypothermia (n = 3). The RI was measured in the anterior cerebral artery and thalamus on days 0, 1, and 4 of life. Magnetic resonance imaging with spectroscopy was performed on day 4. Mean thalamus and anterior cerebral artery RI values in the first 36 hours of life were statistically different in neonates who died (+0.13; P = .019) or developed cerebral palsy (-0.08; P = .003). Thalamic RI values showed stronger associations with serum and spectroscopic lactate values than those in the anterior cerebral artery. The umbilical cord occlusion-with-hypothermia group showed a significant increase in the RI in the thalamus but not the anterior cerebral artery. Resistive index measurements in the thalamus may eventually supplement other bedside measures for predicting outcomes in the HIE population, but further studies need to differentiate the effect of hypothermia from illness severity on thalamic perfusion.

Long-term cognitive effects of uridine treatment in a neonatal rat model of hypoxic-ischemic encephalopathy

Hypoxic-ischemic encephalopathy (HIE), is the most common brain disorder in neonates during the perinatal period, which, to date, can only be managed to some extent by hypothermia. Uridine is the principal circulating pyrimidine in humans which is utilized as a precursor for membrane phospholipid biosynthesis. Uridine has recently been shown to provide clinical benefit in treatment of Alzheimer’s disease due to its involvement in increasing number of brain synapses along with other phospholipid precursors. We previously showed that uridine treatment ameliorated brain damage by reducing apoptosis in a rat model of neonatal HIE. The aim of the present study was to investigate the effects of uridine administration on cognitive functions during periadolescent period in rats subjected to hypoxic-ischemic (HI) brain damage in neonatal period. Male newborn rats were subjected to HI insult on postnatal day 7 (P7) and were injected intraperitoneally with either saline or uridine (500mg/kg) for three consecutive days. Part of pups in each group were sacrificed on P10 to collect brain samples for active Caspase-3 analyses and the remaining pups were raised through P40 to evaluate early reflexes, sensorimotor coordination and learning and memory functions by Negative Geotaxis (NG), Beam Walking (BW) and Morris Water Maze (MWM) tasks, respectively. Confirming our previous findings, we showed that uridine administration reduced apoptotic cell damage on P10. No significant difference was observed between uridine and saline groups in early reflexes or sensorimotor coordination. On the other hand, rats receiving uridine displayed improved learning and memory in MWM during periadolescent period. We conclude that uridine treatment improves learning and memory in the long term by, probably, reducing apoptotic cell death in early newborn period. This is the first study to show beneficial cognitive effects of uridine in rats with brain damage.