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.

Metabolite Biomarkers for Early Ischemic-Hypoxic Encephalopathy: An Experimental Study Using the NeoBase 2 MSMS Kit in a Rat Model.

Hypoxic-ischemic encephalopathy (HIE) is one of the most common causes of childhood disability. Hypothermic therapy is currently the only approved neuroprotective approach. However, early diagnosis of HIE can be challenging, especially in the first hours after birth when the decision to use hypothermic therapy is critical. Distinguishing HIE from other neonatal conditions, such as sepsis, becomes a significant problem in diagnosis. This study explored the utility of a metabolomic-based approach employing the NeoBase 2 MSMS kit to diagnose HIE using dry blood stains in a Rice-Vannucci model of HIE in rats. We evaluated the diagnostic fidelity of this approach in a range between 3 and 6 h after the onset of HIE, including in the context of systemic inflammation and concomitant hypothermic therapy. Discriminant analysis revealed several metabolite patterns associated with HIE. A logistic regression model using glycine levels achieved high diagnostic fidelity with areas under the receiver operating characteristic curve of 0.94 at 3 h and 0.96 at 6 h after the onset of HIE. In addition, orthogonal partial least squares discriminant analysis, which included five metabolites, achieved 100% sensitivity and 80% specificity within 3 h of HIE. These results highlight the significant potential of the NeoBase 2 MSMS kit for the early diagnosis of HIE and could improve patient management and outcomes in this serious illness.

Transplantation of human placental chorionic plate-derived mesenchymal stem cells for repair of neurological damage in neonatal hypoxic-ischemic encephalopathy.

JOURNAL/nrgr/04.03/01300535-202409000-00035/figure1/v/2024-01-16T170235Z/r/image-tiff Neonatal hypoxic-ischemic encephalopathy is often associated with permanent cerebral palsy, neurosensory impairments, and cognitive deficits, and there is no effective treatment for complications related to hypoxic-ischemic encephalopathy. The therapeutic potential of human placental chorionic plate-derived mesenchymal stem cells for various diseases has been explored. However, the potential use of human placental chorionic plate-derived mesenchymal stem cells for the treatment of neonatal hypoxic-ischemic encephalopathy has not yet been investigated. In this study, we injected human placental chorionic plate-derived mesenchymal stem cells into the lateral ventricle of a neonatal hypoxic-ischemic encephalopathy rat model and observed significant improvements in both cognitive and motor function. Protein chip analysis showed that interleukin-3 expression was significantly elevated in neonatal hypoxic-ischemic encephalopathy model rats. Following transplantation of human placental chorionic plate-derived mesenchymal stem cells, interleukin-3 expression was downregulated. To further investigate the role of interleukin-3 in neonatal hypoxic-ischemic encephalopathy, we established an in vitro SH-SY5Y cell model of hypoxic-ischemic injury through oxygen-glucose deprivation and silenced interleukin-3 expression using small interfering RNA. We found that the activity and proliferation of SH-SY5Y cells subjected to oxygen-glucose deprivation were further suppressed by interleukin-3 knockdown. Furthermore, interleukin-3 knockout exacerbated neuronal damage and cognitive and motor function impairment in rat models of hypoxic-ischemic encephalopathy. The findings suggest that transplantation of hpcMSCs ameliorated behavioral impairments in a rat model of hypoxic-ischemic encephalopathy, and this effect was mediated by interleukin-3-dependent neurological function.

Human Placenta Derived Mesenchymal Stem Cells Transplantation Reducing Cellular Apoptosis in Hypoxic-Ischemic Neonatal Rats by Down-Regulating Semaphorin 3A/Neuropilin-1

Neonatal hypoxic-ischemic encephalopathy (HIE) is an abnormal neurological condition caused by hypoxic-ischemic damage during the perinatal period. Human placenta derived mesenchymal stem cells (hPMSCs) have been shown to have protective and reparative effects in various neurological diseases; however, the research on HIE is insufficient. This study aimed to establish a rat model of HIE and transplant hPMSCs through the lateral ventricle after hypoxic-ishcemic (HI) brain damage to observe its protective effects and mechanisms, with a focus on brain apoptosis compared among groups. Differentially expressed apoptosis-related proteins were screened using a rat cytokine array and subsequent verification. Neuropilin-1 (NRP-1) and Semaphorin 3A (Sema 3A) were selected for further investigation. Western blotting was used to quantify the expression of Sema 3A and the proteins related to PI3K/Akt/mTOR signaling pathway. Exogenous Sema 3A was added to evaluate the effects of Sema 3A/NRP-1 on hPMSCs following HI injury. hPMSCs transplantation ameliorated HI-induced pathological changes, reduced apoptosis, and improved long-term neurological prognosis. Furthermore, Sema 3A/NRP-1 was a key regulator in reducing HI-induced apoptosis after hPMSCs transplantation. hPMSCs inhibited the expression of Sema 3A/NRP-1 and activated the PI3K/Akt/mTOR signaling pathway. Additionally, exogenous Sema 3A abolished the protective effects of hPMSCs against HI. In conclusion, hPMSCs transplantation reduced apoptosis and improved long-term neurological prognosis after HI by downregulating Sema 3A/NRP-1 expression and activating the PI3K/Akt/mTOR signaling pathway.

Evaluation of syringin's neuroprotective effect in a model of neonatal hypoxic-ischemic brain injury.

A significant cause of mortality and morbidity in the neonatal era is hypoxic-ischemic encephalopathy (HIE). This study examined the histopathological analysis and neuroprotective impact of syringin (SYR) in an experimental HIE rat model. On the 7th postnatal day, 24 Wistar albino rats were evaluated in 3 groups using the HIE model under gas anesthesia. In the experiment, Group A received 10 mg/kg SYR plus dimethyl sulfoxide (DMSO), Group B received DMSO only, and Group C served as a sham group. Immunohistochemical techniques were used to assess apoptotic cell measurement and proinflammatory cytokines (TNF-α and IL-1β primary antibodies). Rats suffering from hypoxic-ischemic brain damage had their apoptosis assessed. The SYR and sham groups had statistically fewer cells undergoing apoptosis (p < 0.001). There was no difference between the groups in terms of IL-1β and TNF-α during immunohistochemical staining. Neuronal degeneration was significantly lower in the histological evaluation of the hippocampus in the SYR group (p = 0.01). A statistically significant difference (p = 0.01) was observed between the SYR and the control groups regarding pericellular and perivascular edema. SYR reduced apoptosis, perivascular and pericellular edema, and neuronal degeneration in rat cerebral tissue. These results raise the possibility that SYR may have a neuroprotective effect on the harm brought on by HIE. This is the first investigation of SYR's function within the HIE paradigm.

In situ analysis of metabolic changes under hypoxic-ischemic encephalopathy via MALDI mass spectrometry imaging

Hypoxic-ischemic encephalopathy (HIE) is a leading cause of neurological disability and even more serious fetal or neonatal asphyxia death. As the therapeutic time window is limited and timely intervention could have a better prognosis, elucidating the mechanisms underlying HIE and developing novel therapeutic strategies is of great importance. In the present study, 1, 5-Diaminonaphthalene hydrochloride (1, 5-DANHCl) assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) was applied to the neonatal rat model of HIE to investigate metabolic changes during hypoxic-ischemic period. Seventy-three metabolites involved in various metabolic pathways such as glycolysis, tricarboxylic acid (TCA) cycle, nucleoside metabolism, lipid metabolism, oxidative stress and ionic homeostasis demonstrated significant changes. It is worth mentioning that we have detected neutral triglycerides (TGs) that are difficult to ionize and observed their accumulation in the ischemic region, which has been rarely reported in previous studies. The results not only help us discover biomarkers but also provide new insights into its mechanism for us to understand the pathological and physiological processes of the disease.

Ginkgolide B promotes spontaneous recovery and enhances endogenous netrin-1 after neonatal hypoxic-ischemic brain damage.

Perinatal hypoxic-ischemic encephalopathy (HIE) is a condition that can lead to long-term cognitive, motor, and behavioral impairments in newborns. Although brain hypothermia therapy is currently the standard treatment for HIE, it does not provide complete neuroprotection. As a result, there is a need to explore additional therapies to enhance treatment outcomes. This study aims to investigate the potential role of Ginkgolide B (GB) in promoting neuroplasticity and facilitating spontaneous recovery after HIE. In this study, we employed a neonatal rat model of HIE to investigate the effects of GB on spontaneous recovery. GB treatment was initiated 24 h after hypoxia and administered continuously for a duration of 14 days. We evaluated several outcome measures after the treatment period, including spontaneous behavioral recovery and brain repair. Additionally, we quantified the levels of netrin-1 in both plasma and the peri-ischemic zone after the occurrence of HIE. We found that GB treatment significantly facilitated spontaneous behavioral recovery in the HIE pups. Furthermore, cognitive function was restored, and brain tissue repair had a noticeable acceleration. We observed increased cell proliferation in the subventricular, stratum, and subgranular zones. Of particular interest, we observed elevated levels of netrin-1 in both plasma and the ischemic penumbra following GB treatment. Our findings suggest that GB promotes neuroplasticity and enhances spontaneous recovery in newborns affected by HIE. The observed upregulation of netrin-1 may be crucial in mediating these effects. These results highlight the promising potential of GB as a post-HIE therapy, particularly in enhancing spontaneous recovery and improving long-term outcomes.

Differential Effects of Hypothermia and SZR72 on Cerebral Kynurenine and Kynurenic Acid in a Piglet Model of Hypoxic-Ischemic Encephalopathy.

Kynurenic acid (KYNA), an endogenous neuroprotectant with antiexcitotoxic, antioxidant, and anti-inflammatory effects, is synthesized through the tryptophan-kynurenine (KYN) pathway. We investigated whether brain KYN or KYNA levels were affected by asphyxia in a translational piglet model of hypoxic-ischemic encephalopathy (HIE). We also studied brain levels of the putative blood-brain barrier (BBB) permeable neuroprotective KYNA analogue SZR72, and whether SZR72 or therapeutic hypothermia (TH) modified KYN or KYNA levels. KYN, KYNA, and SZR72 levels were determined using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry in five brain regions 24 h after 20 min of asphyxia in vehicle-, SZR72- and TH-treated newborn piglets (n = 6-6-6) and naive controls (n = 4). Endogenous brain KYN levels (median range 311.2-965.6 pmol/g) exceeded KYNA concentrations (4.5-6.0 pmol/g) ~100-fold. Asphyxia significantly increased cerebral KYN and KYNA levels in all regions (1512.0-3273.9 and 16.9-21.2 pmol/g, respectively), increasing the KYN/Tryptophan-, but retaining the KYNA/KYN ratio. SZR72 treatment resulted in very high cerebral SZR72 levels (13.2-33.2 nmol/g); however, KYN and KYNA levels remained similar to those of the vehicle-treated animals. However, TH virtually ameliorated asphyxia-induced elevations in brain KYN and KYNA levels. The present study reports for the first time that the KYN pathway is altered during HIE development in the piglet. SZR72 readily crosses the BBB in piglets but fails to affect cerebral KYNA levels. Beneficial effects of TH may include restoration of the tryptophan metabolism to pre-asphyxia levels.

Farrerol Alleviates Hypoxic-Ischemic Encephalopathy by Inhibiting Ferroptosis in Neonatal Rats via the Nrf2 Pathway.

Farrerol (FA) is a traditional Chinese herbal medicine known for its anti-inflammatory and anti-oxidative properties in various diseases. Ferroptosis is an iron-dependent oxidative stress-induced cell death. It is characterized by lipid peroxidation and glutathione depletion and is involved in neuronal injury. However, the role of FA in inhibiting ferroptosis in hypoxic-ischemic encephalopathy (HIE) and its underlying mechanisms are not yet completely elucidated. This study aimed to investigate whether FA could mediate ferroptosis and explore its function and molecular mechanism in HIE. A neonatal rat model of HIE was used, and rats were treated with FA, ML385 (a specific inhibitor of nuclear factor erythroid 2-related factor 2 [Nrf2]), or a combination of both. Neurological deficits, infarction volume, brain water content, pathological changes, and iron ion accumulation in the brain tissues were measured using the Zea-Longa scoring system and triphenyl tetrazolium chloride (TTC), hematoxylin-eosin (HE), and Perls' staining. The expression levels of GSH-Px, MDA, SOD, and ROS in brain tissues were also evaluated. Western blot analysis was performed to analyze the expression of the Nrf2 pathway and ferroptosis-related proteins. The results showed that FA administration significantly reduced neuronal damage, infarct volume, cerebral edema, and iron ion accumulation and inhibited MDA and ROS levels while promoting GSH-Px and SOD levels. FA also increased the expression levels of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), Nrf2, and HO-1. Moreover, the combination of ML385 and FA in HIE abolished the FA protective effects. Therefore, the study concludes that FA exerts a neuroprotective effect after HIE by inhibiting oxidative stress and ferroptosis via the Nrf2 signaling pathway.

Effect of ligustrazine on hypoxic-ischemic encephalopathy in neonatal rats by regulating autophagy through the PINK1/Parkin pathway

To study the effect of ligustrazine injection on mitophagy in neonatal rats with hypoxic-ischemic encephalopathy (HIE) and its molecular mechanism. Neonatal Sprague-Dawley rats, aged 7 days, were randomly divided into a sham-operation group with 8 rats, a model group with 12 rats, and a ligustrazine group with 12 rats. The rats in the model group and the ligustrazine group were used to establish a neonatal rat model of HIE by ligation of the left common carotid artery followed by hypoxia treatment, and blood vessels were exposed without any other treatment for the rats in the sham-operation group. The rats in the ligustrazine group were intraperitoneally injected with ligustrazine (20 mg/kg) daily after hypoxia-ischemia, and those in the sham-operation group and the model group were intraperitoneally injected with an equal volume of normal saline daily. Samples were collected after 7 days of treatment. Hematoxylin and eosin staining and Nissl staining were used to observe the pathological changes of neurons in brain tissue; immunohistochemical staining was used to observe the positive expression of PINK1 and Parkin in the hippocampus and cortex; TUNEL staining was used to measure neuronal apoptosis; Western blotting was used to measure the expression levels of the mitophagy pathway proteins PINK1 and Parkin and the autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), and ubiquitin-binding protein (P62). Compared with the sham-operation group, the model group had a significant reduction in the number of neurons, an increase in intercellular space, loose arrangement, lipid vacuolization, and a reduction in Nissl bodies. The increased positive expression of PINK1 and Parkin, apoptosis rate of neurons, and protein expression levels of PINK1, Parkin, Beclin1 and LC3 (P<0.05) and the decreased protein expression level of P62 in the hippocampus were also observed in the model group (P<0.05). Compared with the model group, the ligustrazine group had a significant increase in the number of neurons with ordered arrangement and an increase in Nissl bodies, significant reductions in the positive expression of PINK1 and Parkin, the apoptosis rate of neurons, and the protein expression levels of PINK1, Parkin, Beclin1, and LC3 (P<0.05), and a significant increase in the protein expression level of P62 (P<0.05). Ligustrazine can alleviate hypoxic-ischemic brain damage and inhibit neuronal apoptosis in neonatal rats to a certain extent, possibly by inhibiting PINK1/Parkin-mediated autophagy.

Current analysis of hypoxic-ischemic encephalopathy research issues and future treatment modalities.

Hypoxic-ischemic encephalopathy (HIE) is the leading cause of long-term neurological disability in neonates and adults. Through bibliometric analysis, we analyzed the current research on HIE in various countries, institutions, and authors. At the same time, we extensively summarized the animal HIE models and modeling methods. There are various opinions on the neuroprotective treatment of HIE, and the main therapy in clinical is therapeutic hypothermia, although its efficacy remains to be investigated. Therefore, in this study, we discussed the progress of neural circuits, injured brain tissue, and neural circuits-related technologies, providing new ideas for the treatment and prognosis management of HIE with the combination of neuroendocrine and neuroprotection.

LINC00938 alleviates hypoxia ischemia encephalopathy induced neonatal brain injury by regulating oxidative stress and inhibiting JNK/p38 MAPK signaling pathway

Hypoxic-ischemic encephalopathy (HIE) is an important factor leading to permanent damage of central nervous system (CNS) and even neonatal death. Long non-coding RNAs (lncRNAs) has been shown to get involved in the pathogenesis of nervous system diseases. LINC00938 is an intergenic lncRNA which is reported to be involved in neurodegenerative disease. However, the potential role of LINC00938 in nerve injury of neonatal HIE is undetermined. Here, we found that the expression of LINC00938 in the whole blood of neonates with HIE was downregulated compared with the non-HIE group. Functional study revealed that the expression of LINC00938 was significantly decreased in oxygen-glucose deprivation (OGD)-induced SH-SY5Y. Knockdown of LINC00938 induced the neural cell apoptosis by increased the protein level of Bax, Cleaved-Caspase3 and decreased the expression of Bcl-2. In addition, overexpression of LINC00938 prevented the apoptosis of SH-SY5Y from OGD injury. RNA-seq analysis showed that MAPK signaling was involved in the anti-apoptosis function of LINC00938. LINC00938 knockdown induced the activation of c-Jun-N-terminal kinase (JNK), p38 mitogen-activated protein kinase, and inhibited the activation of ERK signaling. However, LINC00938 play neuroprotective role in OGD-induced SH-SY5Y by suppression the phosphorylation of JNK and p38 MAPK rather than regulation of ERK signaling pathway. Further analyses illustrated that the cell apoptosis of neuronal cell was dependent on the elevation of reactive oxygen species (ROS) and result in mitochondria dysfunction in LINC00938 knockdown SH-SY5Y. Pretreated with ROS inhibitor N-acetylcysteine amide (NACA) dramatically suppressed LINC00938 knockdown induced oxidative stress and mitochondria dysfunction which induced cell apoptosis. In addition, NACA treatment significantly reduced the expression of p-JNK and p-p38 in OGD-induced SH-SY5Y. Furthermore, overexpression of LINC00938 displayed a notably neuroprotective effect by suppress central nervous system cell apoptosis via alleviating oxidative stress in CoCl2-induced hypoxic HIE model of zebrafish. Taken together, these results suggested that LINC00938 can act as a neuroprotective factor to inhibit oxidative stress and apoptosis of CNS under HIE conditions.

Surfactant protein a attenuates generalized and localized neuroinflammation in neonatal mice.

Surfactant protein A (SP-A) has important roles in innate immunity and modulation of pulmonary and extrapulmonary inflammation. Given SP-A has been detected in rat and human brain, we sought to determine if SP-A has a role in modulating inflammation in the neonatal mouse brain. Neonatal wildtype (WT) and SP-A-deficient (SP-A-/-) mice were subjected to three models of brain inflammation: systemic sepsis, intraventricular hemorrhage (IVH) and hypoxic-ischemic encephalopathy (HIE). Following each intervention, RNA was isolated from brain tissue and expression of cytokine and SP-A mRNA was determined by real-time quantitative RT-PCR analysis. In the sepsis model, expression of most cytokine mRNAs was significantly increased in brains of WT and SP-A-/- mice with significantly greater expression of all cytokine mRNA levels in SP-A-/- mice compared to WT. In the IVH model, expression of all cytokine mRNAs was significantly increased in WT and SP-A-/- mice and levels of most cytokine mRNAs were significantly increased in SP-A-/- mice compared to WT. In the HIE model, only TNF-α mRNA levels were significantly increased in WT brain tissue while all pro-inflammtory cytokine mRNAs were significantly increased in SP-A-/- mice, and all pro-inflammatory cytokine mRNA levels were significantly higher in SP-A-/- mice compared to WT. SP-A mRNA was not detectable in brain tissue of adult WT mice nor in WT neonates subjected to these models. These results suggest that SP-A-/- neonatal mice subjected to models of neuroinflammation are more susceptible to both generalized and localized neuroinflammation compared to WT mice, thus supporting the hypothesis that SP-A attenuates inflammation in neonatal mouse brain.

Research progress of neonatal hypoxic-ischemic encephalopathy in nonhuman primate models.

Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the important complications of neonatal asphyxia, which not only leads to neurological disability but also seriously threatens the life of neonates. Over the years, animal models of HIE have been a research hotspot to find ways to cope with HIE and thereby reduce the risk of neonatal death or disability in moderate-to-severe HIE. By reviewing the literature related to HIE over the years, it was found that nonhuman primates share a high degree of homology with human gross neural anatomy. The basic data on nonhuman primates are not yet complete, so it is urgent to mine and develop new nonhuman primate model data. In recent years, the research on nonhuman primate HIE models has been gradually enriched and the content is more novel. Therefore, the purpose of this review is to further summarize the methods for establishing the nonhuman primate HIE model and to better elucidate the relevance of the nonhuman primate model to humans by observing the behavioral manifestations, neuropathology, and a series of biomarkers of HIE in primates HIE. Finally, the most popular and desirable treatments studied in nonhuman primate models in the past 5 years are summarized.

Downregulation of TRIM27 alleviates hypoxic-ischemic encephalopathy through inhibiting inflammation and microglia cell activation by regulating STAT3/HMGB1 axis

TRIM27 expression was increased in the Parkinson's disease (PD), and knockdown of TRIM27 in PC12 cells significantly inhibited cell apoptosis, indicating that downregulation of TRIM27 exerts a neuroprotective effect. Herein, we investigated TRIM27 role in hypoxic-ischemic encephalopathy (HIE) and the underlying mechanisms. HIE models were constructed in newborn rats using hypoxic ischemic (HI) treatment and PC-12/BV2 cells with oxygen glucose deprivation (OGD), respectively. The results demonstrated that TRIM27 expression was increased in the brain tissues of HIE rats and OGD-treated PC-12/BV2 cells. Downregulation of TRIM27 reduced the brain infarct volume, inflammatory factor levels and brain injury, as well as decreased the number of M1 subtype of microglia cells while increased the number of M2 microglia cells. Moreover, deletion of TRIM27 expression inhibited the expression of p-STAT3, p-NF-κB and HMGB1 in vivo and in vitro. In addition, overexpression of HMGB1 impaired the effects of TRIM27 downregulation on improving OGD-induced cell viability, inhibiting inflammatory reactions and microglia activation. Collectively, this study revealed that TRIM27 was overexpressed in HIE, and downregulation of TRIM27 could alleviate HI-induced brain injury through repressing inflammation and microglia cell activation via the STAT3/HMGB1 axis.

N-acetylserotonin inhibits oxidized mitochondrial DNA-induced neuroinflammation by activating the AMPK/PGC-1α/TFAM pathway in neonatal hypoxic-ischemic brain injury model

Hypoxic-ischemic encephalopathy (HIE) is a substantial cause of irreversible nerve injury in newborns. As the endogenous precursor of melatonin, N-acetylserotonin (NAS) can cross the blood–brain barrier and exert neuroprotective effects. Evidence shows that NAS exerts a neuroprotective role in ischemic brain injury by inhibiting the mitochondrial death pathway. This study investigates whether NAS can play a neuroprotective role in the HIE model and explores its underlying molecular mechanism. In vivo, postnatal day 7 (P7) Sprague-Dawley rats were used to construct the HIE rat model. Significant improvement in short-term neurobehavioral impairment, cerebral infarction volume decrease, and neuronal injury mitigation was observed with NAS treatment after hypoxic-ischemic (HI) injury. In addition, we validated that NAS suppressed NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome activation and NLRP3 inflammasome-related protein expression in the HIE model. Furthermore, we demonstrate that NAS reduced mitochondrial membrane potential loss, oxidative damage to mitochondrial DNA (mtDNA), cytosolic mtDNA copy number and activation of its downstream cyclic guanosine monophosphate adenylate synthase (cGAS)/ stimulator of interferon genes (STING) pathway. Additionally, we further verified the activation of NAS on the AMP-activated protein kinase (AMPK)/ peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α)/ mitochondrial transcription factor A (TFAM) pathway and found that inhibition of AMPK could increase cytosolic mtDNA copy number and NLRP3 inflammasome-related protein expression while decreasing STING protein expression. Our study demonstrated that NAS inhibits oxidative mtDNA-induced neuroinflammation, which is partially associated with AMPK/PGC-1α/TFAM pathway activation in the HIE model.

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.

Reperfusion after hypoxia-ischemia exacerbates brain injury with compensatory activation of the anti- ferroptosis system: based on a novel rat model.

Hypoxic-ischemic encephalopathy, which predisposes to neonatal death and neurological sequelae, has a high morbidity, but there is still a lack of effective prevention and treatment in clinical practice. To better understand the pathophysiological mechanism underlying hypoxic-ischemic encephalopathy, in this study we compared hypoxic-ischemic reperfusion brain injury and simple hypoxic-ischemic brain injury in neonatal rats. First, based on the conventional Rice-Vannucci model of hypoxic-ischemic encephalopathy, we established a rat model of hypoxic-ischemic reperfusion brain injury by creating a common carotid artery muscle bridge. Then we performed tandem mass tag-based proteomic analysis to identify differentially expressed proteins between the hypoxic-ischemic reperfusion brain injury model and the conventional Rice-Vannucci model and found that the majority were mitochondrial proteins. We also performed transmission electron microscopy and found typical characteristics of ferroptosis, including mitochondrial shrinkage, ruptured mitochondrial membranes, and reduced or absent mitochondrial cristae. Further, both rat models showed high levels of glial fibrillary acidic protein and low levels of myelin basic protein, which are biological indicators of hypoxic-ischemic brain injury and indicate similar degrees of damage. Finally, we found that ferroptosis-related Ferritin (Fth1) and glutathione peroxidase 4 were expressed at higher levels in the brain tissue of rats with hypoxic-ischemic reperfusion brain injury than in rats with simple hypoxic-ischemic brain injury. Based on these results, it appears that the rat model of hypoxic-ischemic reperfusion brain injury is more closely related to the pathophysiology of clinical reperfusion. Reperfusion not only aggravates hypoxic-ischemic brain injury but also activates the anti-ferroptosis system.

Neonatal hypoxic ischemic encephalopathy increases acute kidney injury urinary biomarkers in a rat model.

Hypoxic ischemic encephalopathy (HIE) is associated with acute kidney injury (AKI) in neonates with birth asphyxia. This study aimed to utilize urinary biomarkers to characterize AKI in an established neonatal rat model of HIE. Day 7 Sprague-Dawley rat pups underwent HIE using the Rice-Vannucci model (unilateral carotid ligation followed by 120 mins of 8% oxygen). Controls included no surgery and sham surgery. Weights and urine for biomarkers (NGAL, osteopontin, KIM-1, albumin) were collected the day prior, daily for 3 days post-intervention, and at sacrifice day 14. Kidneys and brains were processed for histology. HIE pups displayed histological evidence of kidney injury including damage to the proximal tubules, consistent with resolving acute tubular necrosis, and had significantly elevated urinary levels of NGAL and albumin compared to sham or controls 1-day post-insult that elevated for 3 days. KIM-1 significantly increased for 2 days post-HIE. HIE did not significantly alter osteopontin levels. Seven days post-start of experiment, controls were 81.2% above starting weight compared to 52.1% in HIE pups. NGAL and albumin levels inversely correlated with body weight following HIE injury. The AKI produced by the Rice-Vannucci HIE model is detectable by urinary biomarkers, which can be used for future studies of treatments to reduce kidney injury.

Abstract 13344: Endothelin B Receptor Agonist, Sovateltide, Promotes Neural Regeneration by Improving Mitochondria Function in a Neonatal Rat Model of Hypoxic-Ischemic Encephalopathy

Introduction: Our preliminary studies have indicated the role of Endothelin B receptors (ETBR) stimulation in decreasing oxidative stress and cell death after sovateltide treatment following hypoxic-ischemic encephalopathy (HIE) in rat pups, which suggests the involvement of ETBR in neonatal hypoxic-ischemic injury. However, its mechanism of action is not yet established. Therefore, this study investigated the effect of sovateltide on mitochondrial dysfunction and neurogenesis in a neonatal rat model of HIE. Hypothesis : Sovateltide protects neuronal survival via preserving mitochondrial activity. Methods: Sprague-Dawley male and female rat pups grouped separately, were divided into 5 different subgroups (1) Control; (2) HIE + Vehicle; (3) HIE + Hypothermia; (4) HIE + sovateltide; and (5) HIE + sovateltide + hypothermia. HIE was induced by ligating the right carotid artery on a postnatal day (PND) 7, followed by hypoxia for 120 min and hypothermia (32-34°C) for 5 h. On PND 7, sovateltide (5 μg/kg, ICV) was injected after hypoxic-ischemic injury. Pups were euthanized on PND 10. Brains were isolated for analysis of markers for mitochondrial fission and fusion as well as neurogenesis in rat brain tissues using western blots. Results : Animals receiving sovateltide showed a significantly increased expression of neuronal differentiation marker, NeuroD1 (p&lt;0.0001) and DoubleCortin (p&lt;0.001) along with neural marker for mature neurons, NeuN (p&lt;0.001) compared to vehicle. Additionally, an upregulation (p&lt;0.0001) of mitochondrial fusion-related proteins, Mfn2, and downregulation of fission proteins, Drp1, in the sovateltide group compared to the vehicle was observed. Higher neuronal progenitor cell differentiation along with better mitochondrial biogenesis in the brain of sovateltide alone or as an adjunct to hypothermia in rat pups was noted. Similar findings were obtained in both male and female rats. Conclusion : Stimulation of ETBR by sovateltide promotes neural regeneration via maintaining mitochondrial dynamics, indicating a potential neuroprotective effect and may, therefore, offer a legitimate therapeutic target for the treatment of neonatal HI brain injury.