Pathogenesis Of Hypoxic-ischemic Encephalopathy Research Articles

The association between plasma IgG N-glycosylation and neonatal hypoxic-ischemic encephalopathy: a case-control study.

Hypoxic-ischemic encephalopathy (HIE) is one of severe neonatal brain injuries, resulting from inflammation and the immune response after perinatal hypoxia and ischemia. IgG N-glycosylation plays a crucial role in various inflammatory diseases through mediating the balance between anti-inflammatory and pro-inflammatory responses. This study aimed to explore the effect of IgG N-glycosylation on the development of HIE. This case-control study included 53 HIE patients and 57 control neonates. An ultrahigh-performance liquid chromatography (UPLC) method was used to determine the features of the plasma IgG N-glycans, by which 24 initial glycan peaks (GPs) were quantified. Multivariate logistic regression was used to examine the association between initial glycans and HIE, by which the significant parameters were used to develop a diagnostic model. Though receiver operating characteristic (ROC) curves, area under the curve (AUC) and 95% confidence interval (CI) were calculated to assess the performance of the diagnostic model. There were significant differences in 11 initial glycans between the patient and control groups. The levels of fucosylated and galactosylated glycans were significantly lower in HIE patients than in control individuals, while sialylated glycans were higher in HIE patients (p < 0.05). A prediction model was developed using three initial IgG N-glycans and fetal distress, low birth weight, and globulin. The ROC analysis showed that this model was able to discriminate between HIE patients and healthy individuals [AUC = 0.798, 95% CI: (0.716-0.880)]. IgG N-glycosylation may play a role in the pathogenesis of HIE. Plasma IgG N-glycans are potential noninvasive biomarkers for screening individuals at high risk of HIE.

Carthamin yellow attenuates brain injury in a neonatal rat model of ischemic-hypoxic encephalopathy by inhibiting neuronal ferroptosis in the hippocampus.

Hypoxic-ischemic encephalopathy (HIE) is a common neurological disorder characterized by ischemia and hypoxia in the perinatal period, which seriously affects the growth and development of newborns. To date, there is no specific drug for the treatment of HIE. Previous studies have shown that ferroptosis plays an important role in the pathogenesis of HIE. Carthamin yellow (CY) is believed to have antioxidant and anti-inflammatory effects. However, no studies have reported the role of CY in ferroptosis in HIE in vivo until now. The aim of this study was to investigate the effect and mechanism of CY on HIE in vivo and to provide an experimental basis for the clinical treatment of HIE. The results demonstrated that CY increased the expression of NeuN in the neonatal rat hypoxic-ischemic brain damage (HIBD) model. Further exploration revealed that CY increased the expression of glutathione peroxidase 4 and ferritin heavy chain 1 while it decreased the expression of PTGS2 and ACSL2. Moreover, CY decreased malondialdehyde expression and increased superoxide dismutase and glutathione expression in vivo. The findings also indicated that CY downregulated the expression of Nrf2 and Keap-1. In conclusion, this study demonstrated that CY attenuated brain injury in an experimental HIBD model, potentially by alleviating hippocampal neuronal ferroptosis through inhibition of the Nrf2/Keap-1 signaling pathway. These findings provide a novel therapeutic strategy for the clinical treatment of HIE.

Atorvastatin Promotes Pro/anti-inflammatory Phenotypic Transformation of Microglia via Wnt/β-catenin Pathway in Hypoxic-Ischemic Neonatal Rats.

Inflammatory reaction plays a key role in the pathogenesis of hypoxic-ischemic encephalopathy (HIE) in neonates. Microglia are resident innate immune cells in the central nervous system and are profoundly involved in neuroinflammation. Studies have revealed that atorvastatin exerts a neuroprotective effect by regulating neuroinflammation in adult animal models of brain stroke and traumatic brain injury, but its role regarding damage to the developing brain remains unclear. This study aimed to clarify the effect and mechanism of atorvastatin on the regulation of microglia function in neonatal hypoxic-ischemic brain damage (HIBD). The oxygen glucose deprivation (OGD) of microglia and neonatal rat HIBD model was established. Atorvastatin, recombinant sclerostin protein (SOST), and XAV939 (degradation of β-catenin) were administered to OGD microglia and HIBD rats. The pathological changes of brain tissue, cerebral infarction volume, learning and memory ability of rats, pro-inflammatory (CD16+/Iba1+) and anti-inflammatory (CD206+/Iba1+) microglia markers, inflammation-related indicators (Inos, Tnfα, Il6, Arg1, Tgfb, and Mrc1), and Wnt/β-catenin signaling molecules were examined. Atorvastatin reduced OGD-induced pro-inflammatory microglia and pro-inflammatory factors, while increasing anti-inflammatory microglia and anti-inflammatory factors. In vivo, atorvastatin attenuated hypoxia-ischemia (HI)-induced neuroinflammation and brain damage. Mechanistically, atorvastatin decreased SOST expression and activated the Wnt/β-catenin signaling pathway, and the administration of recombinant SOST protein or XAV939 inhibited Wnt/β-catenin signaling and attenuated the anti-inflammatory effect of atorvastatin. Atorvastatin promotes the pro/anti-inflammatory phenotypic transformation of microglia via the Wnt/β-catenin pathway in HI neonatal rats. Atorvastatin may be developed as a potent agent for the treatment of HIE in neonates.

Yap1-Usp14 Axis Inhibits Neuronal Mitophagy During Neonatal Hypoxia-Ischemia Encephalopathy by Regulation of Beclin-1 Ubiquitination in Mouse.

Neonatal hypoxic-ischemic encephalopathy (HIE) that results from perinatal cerebral hypoxia-ischemia has become one of the leading causes of acute mortality and chronic disability in infants and children. Despite that neuronal mitophagy and subsequent clearance of damaged neurons exert protective effect, the pathogenesis of HIE and effective treatment strategies for intervention of HIE remain poorly understood. Here, we report that ubiquitin-specific protease 14 (Usp14, a deubiquitinating enzyme) is closely associated with HIE progression by its negative regulation in neuronal mitophagy in mouse. The expression of Usp14 is elevated in both an oxygen-glucose deprivation (OGD) mouse neuronal cell line culture model in vitro and a HIE mouse model in vivo. Mechanistically, OGD treatment activates Hippo signaling that enhances Yap1 phosphorylation levels at Ser-127 but inhibits Yap1 protein level, which potentiates Usp14 transcription and leads to the downregulated ubiquitination at Lys-63 of Beclin-1, a key molecule in autophagy, resulting in the suppressed neuronal mitophagy, subsequent failure in the clearance of damaged neurons, and finally possible dysregulation in brain functions. Thus, our results provide with Usp14 as a novel target and treatment strategy for intervention of HIE, which may help diagnose and treat HIE in clinic.

CSF neopterin and beta-2-microglobulin as inflammation biomarkers in newborns with hypoxic-ischemic encephalopathy.

Inflammation plays a crucial role in the pathogenesis of hypoxic-ischemic encephalopathy (HIE). The aim of this study was to measure inflammation in HIE through an analysis of CSF neopterin and β2-microglobulin and to study the association with brain injury as shown by MRI findings and neurodevelopmental outcomes. CSF biomarkers were measured in study patients at 12 and 72 h. Brain injury was evaluated by MRI, and neurodevelopmental outcomes were assessed at 2-3 years of life. An adverse outcome was defined as the presence of motor or cognitive impairment. Sixty-nine HIE infants were included. Median values of neopterin and β2-microglobulin paralleled the severity of HIE. Adverse outcomes were associated with early neopterin and β2-microglobulin values, late neopterin values, and the neopterin percentage change between the two samples. A cutoff value of 75% neopterin change predicted adverse outcomes with a specificity of 0.9 and a sensitivity of 0.75. CSF neopterin and β2-microglobulin are elevated in HIE, indicating the activation of inflammation processes. Infants with adverse neurodevelopmental outcomes show higher levels of CSF neopterin and β2-microglobulin. The evolution of neopterin levels provides a better predictive capacity than a single determination. Brain inflammation in newborns with HIE could be measurable through the analysis of CSF neopterin and β2-microglobulin, both of which are associated with neurodevelopmental outcomes. Our study introduces two inflammatory biomarkers for infants with HIE that seem to show a more stable profile and are easier to interpret than cytokines. CSF neopterin and β2-m may become clinical tools to monitor inflammation in HIE and might eventually be helpful in measuring the response to emerging therapies.

Peptidomic Analysis of Neonate Umbilical Cord Blood for the Identification of Endogenous Peptides Involved in Hypoxic-Ischemic Encephalopathy.

Neonatal hypoxic–ischemic encephalopathy (HIE) is a common neurological disorder triggered by perinatal cerebral ischemia and hypoxia. Accumulating evidence has shown that peptides have neuroprotective effects in nerve injury. However, the function of endogenous peptides in the pathogenesis of HIE has not been studied. In the present study, a comparative peptidomic profile was performed in the serum of the human umbilical cord blood with HIE (three patients) and the control group (three health control) by liquid chromatography–mass spectrometry (LC-MS). Our study demonstrated that a total of 49 peptides derived from 25 precursor proteins were differentially expressed in the serum of HIE compared with normal controls, including 33 upregulated peptides and 16 downregulated peptides. Each of the differentially expressed peptides has specific characteristics, including pI, Mw, and cleavage pattern. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that the precursor proteins of differentially expressed peptides participate in the different biological process. Moreover, among the 49 differentially expressed peptides, 21 peptides were identified from the fibrinogen chain family, which plays a role in neurological diseases, suggesting that these peptides may play an important role in maintaining brain health. In conclusion, our results showed a comparative peptidomic profile from human umbilical cord blood of HIE patients and normal controls. These dysregulated peptides may have potentially important functions in umbilical cord blood with HIE and may be involved in the pathogenesis of the HIE.

Therapeutic Hypothermia Inhibits the Classical Complement Pathway in a Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy.

ObjectiveComplement activation is instrumental in the pathogenesis of Hypoxic-ischemic encephalopathy (HIE), a significant cause of neonatal mortality and disability worldwide. Therapeutic hypothermia (HT), the only available treatment for HIE, only modestly improves outcomes. Complement modulation as a therapeutic adjunct to HT has been considered, but is challenging due to the wide-ranging role of the complement system in neuroinflammation, homeostasis and neurogenesis in the developing brain. We sought to identify potential therapeutic targets by measuring the impact of treatment with HT on complement effector expression in neurons and glia in neonatal HIE, with particular emphasis on the interactions between microglia and C1q.MethodsThe Vannucci model was used to induce HIE in term-equivalent rat pups. At P10-12, pups were randomly assigned to three different treatment groups: Sham (control), normothermia (NT), and hypothermia (HT) treatment. Local and systemic complement expression and neuronal apoptosis were measured by ELISA, TUNEL and immunofluorescence labeling, and differences compared between groups.ResultsTreatment with HT is associated with decreased systemic and microglial expression of C1q, decreased systemic C5a levels, and decreased microglial and neuronal deposition of C3 and C9. The effect of HT on cytokines was variable with decreased expression of pro and anti-inflammatory effectors. HT treatment was associated with decreased C1q binding on cells undergoing apoptosis.ConclusionOur data demonstrate the extreme complexity of the immune response in neonatal HIE. We propose modulation of downstream effectors C3a and C5a as a therapeutic adjunct to HT to enhance neuroprotection in the developing brain.

Regulatory Mechanism of MicroRNA-30b on Neonatal Hypoxic-Ischemic Encephalopathy (HIE)

ObjectiveThis study is to investigate the role of microRNA (miR)-30b in the pathogenesis of hypoxic-ischemic encephalopathy (HIE) in neonates. MethodsTotally 26 cases of neonatal HIE were included in this study. The protein expression levels of CD26P and PAI-1 were detected with ELISA. Serum levels of miR-30b and PAI-1 mRNA was measured by quantitative real-time PCR. Human brain microvascular endothelial cells (HBMECs) were cultured under hypoxic condition, and the intracellular expression levels of miR-30b and PAI-1 were evaluated. Dual-luciferase reporter assay was performed to confirm the interaction between miR-30b and PAI-1. ResultsCompared with the control group, both the mRNA and protein expression levels of PAI-1 in the serum were up-regulated in the neonates with HIE, together with up-regulated serum CD26P levels. However, the serum expression level of miR-30b was down-regulated in neonatal HIE. In hypoxia-induced HBMECs, the mRNA and protein expression levels of PAI-1 were significantly up-regulated, while the miR-30b expression level was significantly down-regulated. Dual-luciferase reporter assay showed that PAI-1 was the direct target of miR-30b. ConclusionNeonatal HIE is accompanied with abnormal platelet activation, significantly up-regulated serum PAI-1 expression levels, and down-regulated miR-30b expression. MiR-30b might regulate the disease pathogenesis and immune responses via modulating PAI-1.

Single-nucleotide polymorphism screening and RNA sequencing of key messenger RNAs associated with neonatal hypoxic-ischemia brain damage.

A single-nucleotide polymorphism (SNP) is an alteration in one nucleotide in a certain position within a genome. SNPs are associated with disease susceptibility. However, the influences of SNPs on the pathogenesis of neonatal hypoxic-ischemic brain damage remain elusive. Seven-day-old rats were used to establish a hypoxic ischemic encephalopathy model. SNPs and expression profiles of mRNAs were analyzed in hypoxic ischemic encephalopathy model rats using RNA sequencing. Genes exhibiting SNPs associated with hypoxic ischemic encephalopathy were identified and studied by gene ontology and pathway analysis to identify their possible involvement in the disease mechanism. We identified 89 up-regulated genes containing SNPs that were mainly located on chromosome 1 and 2. Gene ontology analysis indicated that the up-regulated genes containing SNPs are mainly involved in angiogenesis, wound healing and glutamatergic synapse and biological processing of calcium-activated chloride channels. Signaling pathway analysis indicated that the differentially expressed genes play a role in glutamatergic synapses, long-term depression and oxytocin signaling. Moreover, intersection analysis of high throughput screening following PubMed retrieval and RNA sequencing for SNPs showed that CSRNP1, DUSP5 and LRRC25 were most relevant to hypoxic ischemic encephalopathy. Significant up-regulation of genes was confirmed by quantitative real-time polymerase chain reaction analysis of oxygen-glucose-deprived human fetal cortical neurons. Our results indicate that CSRNP1, DUSP5 and LRRC25, containing SNPs, may be involved in the pathogenesis of hypoxic ischemic encephalopathy. These findings indicate a novel direction for further hypoxic ischemic encephalopathy research. This animal study was approved on February 5, 2017 by the Animal Care and Use Committee of Kunming Medical University, Yunnan Province, China (approval No. kmmu2019038). Cerebral tissue collection from a human fetus was approved on September 30, 2015 by the Ethics Committee of Kunming Medical University, China (approval No. 2015-9).

MicroRNA-204 may participate in the pathogenesis of hypoxic-ischemic encephalopathy through targeting KLLN.

Hypoxic-ischemic encephalopathy (HIE) is a common neonatal disease that can lead to high neonatal mortality rates. Previous studies have indicated that microRNAs (miRs) may be involved in the pathogenesis of HIE; however, the specific mechanisms underlying their involvement require further investigation. The aim of the present study was to investigate the roles of miR-204 and its target gene killin p53 regulated DNA replication inhibitor (KLLN) in HIE using rat HIE models. Brain injury was induced by surgery and incubation of hypoxic incubator brain using 10-day-old pup rats. On day 3, rats were sacrificed, and the infarct size of the brain was determined using a tetrazolium chloride assay. Terminal deoxynucleotidyl transferase UTP nick-end labeling staining was performed to detect the cell death rate in the brain tissue. Following this, the brain tissues were collected, and reverse transcription-quantitative polymerase chain reaction, western blot analysis and immunohistochemistry assays were performed to examine the expression levels of miR-204 and KLLN. Furthermore, neurons were cultured and transfected with miR-204 inhibitors or mimics, and the effect of miR-204 on the proliferation and apoptosis of neurons was examined using MTT and flow cytometric assays. Finally, a dual-luciferase reporter assay was performed to confirm whether KLLN is a direct target of miR-204. The expression of miR-204 was significantly downregulated and the expression of KLLN was significantly increased in the brain tissue of HIE rats (P<0.001). In addition, the transfection with miR-204 inhibitors significantly decreased the proliferation rates and significantly increased the apoptosis rate of neurons; however, transfection with miR-204 mimics prompted the opposite results. The dual-luciferase reporter assay also confirmed that KLLN is a direct target of miR-204. Taken together, the results of the present study demonstrated that miR-204 was downregulated in HIE and that miR-204 may serve important roles in the pathogenesis of HIE through targeting KLLN.

Atomoxetine, a selective norepinephrine reuptake inhibitor, improves short-term histological outcomes after hypoxic-ischemic brain injury in the neonatal male rat

BackgroundDespite the recent progress of perinatal medicine, perinatal hypoxic-ischemic (HI) insult remains an important cause of brain injury in neonates, and is pathologically characterized by neuronal loss and the presence of microglia. Neurotransmitters, such as norepinephrine (NE) and glutamate, are involved in the pathogenesis of hypoxic-ischemic encephalopathy via the interaction between neurons and microglia. Although it is well known that the monoamine neurotransmitter NE acts as an anti-inflammatory agent in the brain under pathological conditions, its effects on perinatal HI insult remains elusive. Atomoxetine, a selective NE reuptake inhibitor, has been used clinically for the treatment of attention-deficit hyperactivity disorder in children. Here, we investigated whether the enhancement of endogenous NE by administration of atomoxetine could protect neonates against HI insult by using the neonatal male rat model. We also examined the involvement of microglia in this process. MethodsUnilateral HI brain injury was induced by the combination of left carotid artery dissection followed by ligation and hypoxia (8% O2, 2 h) in postnatal day 7 (P7) male rat pups. The pups were randomized into three groups: the atomoxetine treatment immediately after HI insult, the atomoxetine treatment at 3 h after HI insult, or the vehicle treatment group. The pups were euthanized on P8 and P14, and the brain regions including the cortex, striatum, hippocampus, and thalamus were evaluated by immunohistochemistry. ResultsHI insult resulted in severe brain damage in the ipsilateral hemisphere at P14. Atomoxetine treatment immediately after HI insult significantly increased NE levels in the ipsilateral hemisphere at 1 h after HI insult and reduced the neuronal damage via the increased phosphorylation of cAMP response element-binding protein (pCREB) in all brain regions examined. In addition, the number of microglia was maintained under atomoxetine treatment compared with that of the vehicle treatment group. To determine the involvement of microglia in the process of neuronal loss by HI insult, we further examined the influence of hypoxia on rat primary cultured microglia by the quantitative real-time polymerase chain reaction. Hypoxia did not cause the upregulation of interleukin-1beta (IL-1β) mRNA expression, but decreased the microglial intrinsic nitric oxide synthase (iNOS)/arginase1 mRNA expression ratio. NE treatment further decreased the microglial iNOS/arginase1 mRNA expression ratio. In contrast, no significant neuroprotective effect was observed at P14 when atomoxetine was administered at 3 h after HI insult. ConclusionsThese findings suggested that the enhancement of intrinsic neurotransmitter NE signaling by a selective NE reuptake inhibitor, atomoxetine, reduced the perinatal HI insult brain injury. In addition, atomoxetine treatment was associated with changes of TUNEL, pCREB, and BDNF expression levels, and microglial numbers, morphology, and responses.

Molecular hydrogen alleviates asphyxia-induced neuronal cyclooxygenase-2 expression in newborn pigs.

Cyclooxygenase-2 (COX-2) has an established role in the pathogenesis of hypoxic-ischemic encephalopathy (HIE). In this study we sought to determine whether COX-2 was induced by asphyxia in newborn pigs, and whether neuronal COX-2 levels were affected by H2 treatment. Piglets were subjected to either 8 min of asphyxia or a more severe 20 min of asphyxia followed by H2 treatment (inhaling room air containing 2.1% H2 for 4 h). COX-2 immunohistochemistry was performed on brain samples from surviving piglets 24 h after asphyxia. The percentages of COX-2-immunopositive neurons were determined in cortical and subcortical areas. Only in piglets with more severe HIE, we observed significant, region-specific increases in neuronal COX-2 expression within the parietal and occipital cortices and in the CA3 hippocampal subfield. H2 treatment essentially prevented the increases in COX-2-immunopositive neurons. In the parietal cortex, the attenuation of COX-2 induction was associated with reduced 8'-hydroxy-2'-deoxyguanozine immunoreactivity and retained microglial ramifcation index, which are markers of oxidative stress and neuroinfiammation, respectively. This study demonstrates for the first time that asphyxia elevates neuronal COX-2 expression in a piglet HIE model. Neuronal COX-2 induction may play region-specific roles in brain lesion progression during HIE development, and inhibition of this response may contribute to the antioxidant/anti-infiammatory neuroprotective effects of H2 treatment.

АМІНОКИСЛОТИ В ПАТОГЕНЕЗІ ПЕРИНАТАЛЬНОЇ ГІПОКСИЧНО-ІШЕМІЧНОЇ ЕНЦЕФАЛОПАТІЇ

На долю гіпоксично-ішемічної енцефалопатії (ГІЕ) припадає близько половини всіх перинатальних уражень головного мозку, які є основною причиною інвалідизації та дезадаптації дітей. Роль і взаємодія метаболічних шляхів у патогенезі ГІЕ ясні ще не до кінця; виявлені значні зміни в метаболізмі амінокислот (АК).Мета дослідження – вивчити зміни метаболізму амінокислот при гіпоксично-ішемічній енцелопатії.Матеріали і методи. Було обстежено 117 новонароджених із ГІЕ, з них 60 у гострому періоді (у віці до 1 місяця) і 57 в ранньому відновлювальному періоді гіпоксично-ішемічної енцефалопатії (у віці 1–4 місяці). Усім пацієнтам проводилося дослідження вільних амінокислот крові методом високоефективної рідинної хроматографії.Результати досліджень та їх обговорення. В гострому періоді ГІЕ виявлено підвищення рівнів нейроамінокислот – глутамату (р=0,001), гліцину (р=0,038), також метіоніну (р=0,015) і АК, що задіяні в енергетичному метаболізмі та підтримці постійного рівня глюкози крові: аланін (р=0,001), треонін (р=0,028), амінокислоти з розгалуженим ланцюгом – валін (р=0,01), лейцин (р=0,021); зниження рівнів триптофану (р=0,000) і тирозину (р=0,015), які є попередниками нейромедіаторів. У ранньому відновлювальному періоді ГІЕ, порівняно з гострим, відмічались частіші зміни АК циклу сечовини, аспартату; зниження рівнів валіну.Висновки. Виявлені дані підтверджують, що метаболізм амінокислот займає одне з провідних місць у патогенезі ГІЕ; найбільш часто виявлялися зміни амінокислот, які беруть участь у процесах нейротрансмісії та енергетичному метаболізмі, детоксикації аміаку. Це дає підставу для проведення метаболічної корекції і профілактики ускладнень.

Interleukin-1β, interleukin-6, and tumor necrosis factor-α in the cerebrospinal fluid of term infants with hypoxic-ischemic encephalopathy after postnatal treatment with magnesium sulfate

Proinflammatory cytokines play a role in the pathogenesis of hypoxic-ischemic encephalopathy (HIE). We aimed: 1) to characterize the expression of interleukin-1 β (IL-1 β ), IL-6 and tumor necrosis factor- α (TNF- α ) in the cerebrospinal fluid (CSF) of term infants with HIE, and 2) to determine the impact of a single dose of magnesium sulfate (MgSO 4 ) on their concentrations. We analyzed the samples of CSF that were collected during a randomized controlled trial on the use of MgSO 4 in neonatal asphyxia. Two samples were obtained from each subject; one was obtained on admission and the second sample was collected at 72 hr of life. The grades of HIE in the 47 included newborns were mild ( n = 15), moderate ( n = 17), and severe ( n = 15). After the collection of the first CSF sample, 23 newborns received a single dose of MgSO 4 , whereas the other 24 newborns received an equal volume of normal saline placebo. Concentrations of IL-1 β , IL-6 and TNF- α in the CSF were quantified by enzyme-linked immunosorbent assay in all CSF samples. The initial concentration of IL-1 β and IL-6 correlated with the severity grades of HIE ( P = 004 and P = 0001, respectively) while TNF- α did not. At 72 hr, a significant decline in the concentration of TNF- α (pg/mL) was observed in samples of MgSO 4 -treated infants when compared to the control group (–28 ± 90 vs. 102 ± 404, P = 0.01), whereas the two treatment groups did not differ in IL-1 β or IL-6. Short-term clinical outcomes correlated best with IL-1 β at baseline, IL-6 at baseline and IL-6 at 72 hr. We conclude that cytokine activation, as manifested by increased IL-1 β and IL-6 in the CSF, is positively correlated with the severity of HIE. Treatment with a single dose of MgSO 4 was efficacious in aborting the activation of TNF- α but did not alter the expression of IL-1 β or IL-6.

Functional Polymorphism Of The Interleukin-1beta Gene Promoter Is Associated With Increased Risk For Cerebral Palsy In Mexican Children With Perinatal Hypoxia-Ischemia Antecedents

Background: The genetic polymorphism that contributes to the development of infantile cerebral palsy (ICP) after a perinatal hypoxia-ischemia episode remains unknown. Because IL-1? plays a crucial role in the pathogenesis of hypoxic-ischemic encephalopathy, we evaluated whether the interleukin 1, beta (IL-1?) promoter single-nucleotide polymorphisms (SNPs) correlate with the increased risk for ICP following a perinatal hypoxia-ischemia. We assessed -511 C>T and -31 T>C IL-1? SNPs known to be involved in IL-1? expression. Methods: Genomic DNAs were purified from peripheral leukocytes of 48 ICP patients and 57 healthy children, amplified by using polymerase chain reaction (PCR), and then analyzed by using the restriction-fragment-length polymorphism technique. The SNP genotypes were established using real time PCR and validated with the restriction enzymes AvaI for -511C-T and AluI for -31T-C in Restriction fragment length polymorphism (RFLP) analysis. Results: Allelic frequencies of the IL-1? -511 T carrier in patients were significantly higher when compared with those determined in healthy controls. The -511 TT genotype frequency showed a significant difference [odds ratio = 2.4 (95% confidence interval 1.7-3.5), P = 0.0001, and relative risk = 1.5 (95% confidence interval 1.3-1.7)] between patients and controls. The SNP frequencies of -31 genotypes in cerebral palsy patients were not statistically different from the healthy controls. Conclusions: Mexican children with the homozygous TT mutation in the -511 SNP of the IL-1? gene promoter are 2.4 times more susceptible to develop ICP after suffering perinatal asphyxia than healthy children and the presence of a single allele C could be considered as a genetic protective factor.

5ICCN_027: The effect of phototherapy on oxidative stress and inflammatory response in newborns

Hypoxic ischemic encephalopathy (HIE), despite advances in diagnosis and treatment, is still a major problem which leads to increased morbidity and mortality. Previous studies suggested that inflammatory cycle induced by hypoxia and ischemia play a role in the pathogenesis of HIE. In this study we investigated the relationship between HIE and inflammatory response by measuring IL-6, IL-18 and hsCRP levels in newborn infants with HIE. All term infants who have diagnosed HIE, delivered in our institute between study period were included in the study. Infants were classificated to stage I, stage II and stage III according to clinical findings (Sarnat & Sarnat classification were used). IL-6, IL-18 and hsCRP levels were determined in blood samples; in the first 24 hours (early period), and 48–72 hours (late period) respectively. 30 patients were included in the study. We found that IL-6 levels were decreased and IL-18 and hsCRP were increased in the late period. Increased levels of IL-18 and hsCRP defined as statistically significant (p < 0.01, p < 0.05 respectively). Patients who died had higher levels of IL-18 in the late period. High serum IL-18 and hsCRP levels in the late period are associated with advanced stages of HIE.

5ICCN_026: The IL-6, IL-18 and hsCRP levels of the newborn infants with hypoxic ischemic encephalopathy

Hypoxic ischemic encephalopathy (HIE), despite advances in diagnosis and treatment, is still a major problem which leads to increased morbidity and mortality. Previous studies suggested that inflammatory cycle induced by hypoxia and ischemia play a role in the pathogenesis of HIE. In this study we investigated the relationship between HIE and inflammatory response by measuring IL-6, IL-18 and hsCRP levels in newborn infants with HIE. All term infants who have diagnosed HIE, delivered in our institute between study period were included in the study. Infants were classificated to stage I, stage II and stage III according to clinical findings (Sarnat & Sarnat classification were used). IL-6, IL-18 and hsCRP levels were determined in blood samples; in the first 24 hours (early period), and 48–72 hours (late period) respectively. 30 patients were included in the study. We found that IL-6 levels were decreased and IL-18 and hsCRP were increased in the late period. Increased levels of IL-18 and hsCRP defined as statistically significant (p < 0.01, p < 0.05 respectively). Patients who died had higher levels of IL-18 in the late period. High serum IL-18 and hsCRP levels in the late period are associated with advanced stages of HIE.

Cyclosporine treatment reduces oxygen free radical generation and oxidative stress in the brain of hypoxia-reoxygenated newborn piglets.

Oxygen free radicals have been implicated in the pathogenesis of hypoxic-ischemic encephalopathy. It has previously been shown in traumatic brain injury animal models that treatment with cyclosporine reduces brain injury. However, the potential neuroprotective effect of cyclosporine in asphyxiated neonates has yet to be fully studied. Using an acute newborn swine model of hypoxia-reoxygenation, we evaluated the effects of cyclosporine on the brain, focusing on hydrogen peroxide (H2O2) production and markers of oxidative stress. Piglets (1–4 d, 1.4–2.5 kg) were block-randomized into three hypoxia-reoxygenation experimental groups (2 h hypoxia followed by 4 h reoxygenation)(n = 8/group). At 5 min after reoxygenation, piglets were given either i.v. saline (placebo, controls) or cyclosporine (2.5 or 10 mg/kg i.v. bolus) in a blinded-randomized fashion. An additional sham-operated group (n = 4) underwent no hypoxia-reoxygenation. Systemic hemodynamics, carotid arterial blood flow (transit-time ultrasonic probe), cerebral cortical H2O2 production (electrochemical sensor), cerebral tissue glutathione (ELISA) and cytosolic cytochrome-c (western blot) levels were examined. Hypoxic piglets had cardiogenic shock (cardiac output 40–48% of baseline), hypotension (mean arterial pressure 27–31 mmHg) and acidosis (pH 7.04) at the end of 2 h of hypoxia. Post-resuscitation cyclosporine treatment, particularly the higher dose (10 mg/kg), significantly attenuated the increase in cortical H2O2 concentration during reoxygenation, and was associated with lower cerebral oxidized glutathione levels. Furthermore, cyclosporine treatment significantly attenuated the increase in cortical cytochrome-c and lactate levels. Carotid blood arterial flow was similar among groups during reoxygenation. Conclusively, post-resuscitation administration of cyclosporine significantly attenuates H2O2 production and minimizes oxidative stress in newborn piglets following hypoxia-reoxygenation.

Clinical significance of changes of RBC [Ca2 +]i levels in the neonates with hypoxic-ischemic encephalopathy

Objective To explore the role of RBC[Ca2+]i levels in pathogenesis of hypoxic-ischemic encephalopathy (HIE) in neonates. Methods Twenty-eight neonates with moderate and severe HIE hospitalizeal from Jun. 2002 to Mar. 2006 were enrolled the study. The neonates with HIE were given routine treatment and Nimodipine for 7～10 days. Blood samples were collected before treatment and at 72 hours,7～10 days after treatment respectively. The levels of RBC [Ca2+]i were measured by Fura-2/AM. Twenty healthy full-term neonates were studied as controls. Results (1) The levels of RBC [Ca2+] i in the neonates with moderate and severe HIE were significantly higher than that in control group at every time points( P＜0. 05 ,P＜0.01). (2) the levels of RBC[Ca2+]i in the neonates with moderate and severe HIE peaked at 72 hours after treatment,and were still significantly higher than that of control group at 7～10 days after treatment(P＜0. 05). (3) In the neonates with HIE,RBC[Ca2+ ]i levels correlated positively with the severity of HIE ( r = 0. 447, P＜ 0. 05 ). Conclusion RBC [Ca2+ ] i levels are closely associated with pathogenesis of HIE, and may play an important role in the pathogenesis of HIE. Evaluating RBC [ Ca2+] i levels in neonate after birth may provide clinical clues for the early diagnosis and prognosis assessment of HIE. Key words: Cerebral hypoxia; Cerebral ischemia; RBC[Ca2+]i; Infant,newborn

Expression of T subsets and mIL-2R in peripheral blood of newborns with hypoxic ischemic encephalopathy

Infantile and undifferentiated immune cells in the pathogenesis of neonates with HIE have been studied in recent years. This study was undertaken to observe the expression level of T subsets and membrane interleukin-2 receptor (mIL-2R) in the peripheral blood of newborns with hypoxic ischemic encephalopathy (HIE) and its clinical manifestations. The peripheral blood mononuclear cells (PBMCs) of newborns with HIE and normal controls were isolated by the routine Ficoll-Hypaque method, and the rates of CD3+, CD4+, CD8+, CD4+/CD8+ and mIL-2R induced and not induced by phytohemagglutinin (PHA) were detected by biotin-streptavidin (BSA) at the first, third and seventh day after birth. At the first day after birth, the positive rates of CD3+, CD4+, CD8+, CD4+/CD8+ and mIL-2R induced and not induced by PHA were (37.4+/-6.7)%, (29.4+/-6.9)%, (16.7+/-3.3)%, 1.8+/-0.5, (3.6+/-1.1)% and (20.9+/-4.8)%, respectively. Significant differences were observed between the HIE group and the normal controls (P<0.01-P<0.05). At the third day after birth, the positive rates of CD3+, CD4+, CD8+, CD4+/CD*+ and mIL-2R induced and not induced by PHA were (41.0+/-7.4)%, (35.8+/-6.9)%, (22.6+/-4.5)%, (1.7+/-0.5), (3.9+/-1.2)%, and (22.8+/-5.1)%, respectively. There were significant differences between the HIE group and the normal controls (P<0.05). At the seventh day after birth, the positive rates of CD3+, CD4+, CD8+ were (41.8+/-6.1)%, (36.4+/-5.1)% and (25.6+/-4.3)%, respectively. There was significant difference between the HIE group and the normal controls (P<0.05). The ratio of CD4+/CD8+ and the expression level of mIL-2R induced and not induced by PHA were 1.5+/-0.3, (4.1+/-1.2)% and (23.8+/-5.2)%, respectively. There was no significant difference between the HIE group and the normal controls (P>0.05). Peripheral blood mononuclear cells of newborns are immature and undifferentiated with a very low expression level of surface markers. The changes of cell immunity involve in the pathogenesis of HIE. The disorder of cellular immune function exists in newborns with HIE. Cell immunity and immune regulative response in newborns are gradually improved or mature during the period of growing, facilitating the recovery from brain injury caused by HIE.