Hypoxia-ischemia In Neonatal Rats Research Articles

Inter-alpha Inhibitor Proteins Modulate Microvascular Endothelial Components and Cytokines After Exposure to Hypoxia-Ischemia in Neonatal Rats.

Inter-alpha inhibitor proteins (IAIPs) are neuroprotective and attenuate lipopolysaccharide (LPS)-mediated blood-brain barrier (BBB) disruption in neonatal rodents. We investigated some mechanism(s) fundamental to neuroprotection by IAIPs including changes in cerebral endothelial components and inflammation. Postnatal day-7 rats exposed to sham surgery and placebo or carotid ligation plus 8% FiO2 (90min) were given IAIPs (30 or 60mg/kg) or placebo and were killed 6, 12, 24, or 36h after hypoxia-ischemia (HI). Proteins regulating BBB permeability to leukocytes (vascular cell adhesion molecule 1, VCAM-1), lipid-soluble (P-glycoprotein, PGP), and lipid-insoluble molecules (zonula occludens-1, ZO-1) were measured by immunoblot, and cytokines were measured in serum and cortex. HI resulted in reductions in ZO-1 and increases in VCAM-1, PGP, interferon-γ (IFN-γ), interleukin-12 (IL-12), vascular endothelial growth factor (VEGF), IL-α, and macrophage colony-stimulating factor (M-CSF) in cortex and increases in IL-4, IL-5, IL-10, and granulocyte colony-stimulating factor (G-CSF) in serum. IAIPs attenuated the reductions in ZO-1 and delayed increases in VCAM-1 and PGP in cortex and attenuated increases in cytokines in serum (IL-4, IL-5, IL-10, IFN-γ, G-CSF) and cortex (IL-1α, IL-12, IFN-γ, VEGF, M-CSF) after HI. We conclude that vascular endothelial proteins and cytokines exhibit sequential changes after HI and IAIPs modulate some of these HI-related changes in neonatal rats.

Neuroprotective Effect of Melatonin in a Neonatal Hypoxia-Ischemia Rat Model Is Regulated by the AMPK/mTOR Pathway.

Melatonin has been shown to be neuroprotective in different animal models of neonatal hypoxic-ischemic brain injury. However, its exact molecular mechanism of action remains unknown. Our aim was to prove melatonin's short- and long-term neuroprotection and investigate its role on the AMPK (AMP-activated protein kinase)/mTOR (mammalian target of rapamycin) pathway following neonatal hypoxic-ischemic brain injury. Seven-day-old Wistar rat pups were exposed to hypoxia-ischemia, followed by melatonin or vehicle treatment. Detailed analysis of the AMPK/mTOR/autophagy pathway, short- and long-term neuroprotection, myelination, and oligodendrogenesis was performed at different time points. At 7 days after hypoxia-ischemia, melatonin-treated animals showed a significant decrease in tissue loss, increased oligodendrogenesis, and myelination. Long-term neurobehavioral results showed significant motor improvement following melatonin treatment. Molecular pathway analysis showed a decrease in the AMPK expression, with a significant increase at mTOR's downstream substrates, and a significant decrease at the autophagy marker levels in the melatonin group compared with the vehicle group. Melatonin treatment reduced brain area loss and promoted oligodendrogenesis with a clear improvement of motor function. We found that melatonin associated neuroprotection is regulated via the AMPK/mTOR/autophagy pathway. Considering the beneficial effects of melatonin and the results of our study, melatonin seems to be an optimal candidate for the treatment of newborns with hypoxic-ischemic brain injury in high- as well as in low- and middle-income countries.

Dimethyl Fumarate Strongly Ameliorates Gray and White Matter Brain Injury and Modulates Glial Activation after Severe Hypoxia-Ischemia in Neonatal Rats.

Neonatal hypoxia-ischemia is a major cause of infant death and disability. The only clinically accepted treatment is therapeutic hypothermia; however, cooling is less effective in the most severely encephalopathic infants. Here, we wanted to test the neuroprotective effect of the antioxidant dimethyl fumarate after severe hypoxia-ischemia in neonatal rats. We used a modified Rice-Vannucci model to generate severe hypoxic-ischemic brain damage in day 7 postnatal rats, which were randomized into four experimental groups: Sham, Sham + DMF, non-treated HI, and HI + DMF. We analyzed brain tissue loss, global and regional (cortex and hippocampus) neuropathological scores, white matter injury, and microglial and astroglial reactivity. Compared to non-treated HI animals, HI + DMF pups showed a reduced brain area loss (p = 0.0031), an improved neuropathological score (p = 0.0016), reduced white matter injuries by preserving myelin tracts (p < 0.001), and diminished astroglial (p < 0.001) and microglial (p < 0.01) activation. After severe hypoxia-ischemia in neonatal rats, DMF induced a strong neuroprotective response, reducing cerebral infarction, gray and white matter damage, and astroglial and microglial activation. Although further molecular studies are needed and its translation to human babies would need to evaluate the molecule in piglets or lambs, DMF may be a potential treatment against neonatal encephalopathy.

Genistein-3′-sodium sulfonate enhances neurological function in neonatal rats with hypoxia-ischemia during the recovery period

Hypoxic-ischemic (HI) can cause neonatal brain damage leading to disability. Patients with HI experience long-term neurological issues impacting quality of life. Limited clinical treatments are available despite extensive research on HI's molecular mechanisms. Genistein-3′-sodium sulfonate (GSS), a phytoestrogen, has been found to improve acute brain injury in neonatal rats caused by hypoxic-ischemia, but its potential for chronic stage neurological recovery in HI is unknown. HI neonatal rats were treated with 1 mg/kg GSS once a day for 21 days. Then, a series of behavioral experiments was performed to evaluate the learning, memory, cognition, anxiety level and depression-like behaviors of the rats. GSS treatment reduced neuronal loss, enhanced learning, memory and cognitive function while also alleviated anxiety and depression-like behaviors in HI rats during the recovery period. These findings indicated that GSS exerted enhance neurological function in HI rats during the chronic stage, prompting further research on how it works to potentially develop new therapies.

Neuroprotective Effect of Clemastine Improved Oligodendrocyte Proliferation through the MAPK/ERK Pathway in a Neonatal Hypoxia Ischemia Rat Model.

Neonatal hypoxic-ischemic encephalopathy is the most common cause of long-term disability in term neonates, and white matter injury is the primary cause of cerebral palsy. Therapies that focus on the neuroprotection of myelination and oligodendrocyte proliferation could potentially ameliorate long-lasting neurological impairments after hypoxic-ischemic encephalopathy. Clemastine, a histamine H1 antagonist, has been shown to exert neuroprotective effects in multiple sclerosis and spinal cord injury by promoting oligodendrogenesis and re-myelination. In this study, we demonstrated the neuroprotective effects of clemastine in our rat model of neonatal hypoxic-ischemic brain injury. Animals received a single intraperitoneal injection of either vehicle or clemastine (10 mg/kg) for 6 consecutive days. Our results showed a significant reduction in white matter loss after treatment, with a clear effect of clemastine on oligodendrocytes, showing a significant increase in the number of Olig2+ cells. We characterized the MAPK/ERK pathway as a potential mechanistic pathway underlying the neuroprotective effects of clemastine. Altogether, our results demonstrate that clemastine is a potential compound for the treatment of hypoxic-ischemic encephalopathy, with a clear neuroprotective effect on white matter injury by promoting oligodendrogenesis.

Activation of the CD200/CD200R1 axis improves cognitive impairment by enhancing hippocampal neurogenesis via suppression of M1 microglial polarization and neuroinflammation in hypoxic-ischemic neonatal rats

Following hypoxic-ischemic brain damage (HIBD), there is a decline in cognitive function; however, there are no effective treatment strategies for this condition in neonates. This study aimed to evaluate the role of the cluster of differentiation 200 (CD200)/CD200R1 axis in cognitive function following HIBD using an established model of HIBD in postnatal day 7 rats. Western blotting analysis was conducted to evaluate the protein expression levels of CD200, CD200R1, proteins associated with the PI3K/Akt-NF-κB pathway, and inflammatory factors such as TNF-α, IL-1β, and IL-6 in the hippocampus. Additionally, double-immunofluorescence labeling was utilized to evaluate M1 microglial polarization and neurogenesis in the hippocampus. To assess the learning and memory function of the experimental rats, the Morris water maze (MWM) test was conducted. HIBDleads to a decrease in the expression of CD200 and CD200R1 proteins in the neonatal rat hippocampus, while simultaneously increasing the expression of TNF-α, IL-6, and IL-1β proteins, ultimately resulting in cognitive impairment. The administration of CD200Fc, a fusion protein of CD200, was found to enhance the expression of p-PI3K and p-Akt, but reduce the expression of p-NF-κB. Additionally, CD200Fc inhibited M1 polarization of microglia, reduced neuroinflammation, improved hippocampal neurogenesis, and mitigated cognitive impairment caused by HIBD in neonatal rats. In contrast, blocking the interaction between CD200 and CD200R1 with the anti-CD200R1 antibody (CD200R1 Ab) exerted the opposite effect. Furthermore, the PI3K specific activator, 740Y-P, significantly increased the expression of p-PI3K and p-Akt, but reduced p-NF-κB expression. It also inhibited M1 polarization of microglia, reduced neuroinflammation, and improved hippocampal neurogenesis and cognitive function in neonatal rats with HIBD. Our findings illustrate that activation of the CD200/CD200R1 axis inhibits the NF-κB-mediated M1 polarization of microglia to improve HIBD-induced cognitive impairment and hippocampal neurogenesis disorder via the PI3K/Akt signaling pathway.

N6-methyladenosine demethylase FTO regulates synaptic and cognitive impairment by destabilizing PTEN mRNA in hypoxic-ischemic neonatal rats

Hypoxic-ischemic brain damage (HIBD) can result in significant global rates of neonatal death or permanent neurological disability. N6-methyladenosine (m6A) modification of RNA influences fundamental aspects of RNA metabolism, and m6A dysregulation is implicated in various neurological diseases. However, the biological roles and clinical significance of m6A in HIBD remain unclear. We currently evaluated the effect of HIBD on cerebral m6A methylation in RNAs in neonatal rats. The m6A dot blot assay showed a global augmentation in RNA m6A methylation post-HI. Herein, we also report on demethylase FTO, which is markedly downregulated in the hippocampus and is the main factor involved with aberrant m6A modification following HI. By conducting a comprehensive analysis of RNA-seq data and m6A microarray results, we found that transcripts with m6A modifications were more highly expressed overall than transcripts without m6A modifications. The overexpression of FTO resulted in the promotion of Akt/mTOR pathway hyperactivation, while simultaneously inhibiting autophagic function. This is carried out by the demethylation activity of FTO, which selectively demethylates transcripts of phosphatase and tensin homolog (PTEN), thus promoting its degradation and reduced protein expression after HI. Moreover, the synaptic and neurocognitive disorders induced by HI were effectively reversed through the overexpression of FTO in the hippocampus. Cumulatively, these findings demonstrate the functional importance of FTO-dependent hippocampal m6A methylome in cognitive function and provides novel mechanistic insights into the therapeutic potentials of FTO in neonatal HIBD.

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.

Hypoxia ischemia results in blood brain barrier damage via AKT/GSK-3β/CREB pathway in neonatal rats

Previous studies have showed that the permeability of blood brain barrier (BBB) increased after hypoxia ischemia (HI). The current research uncovered the mechanism of altered BBB permeability after hypoxic-ischemic brain damage (HIBD) through AKT/GSK-3β/CREB signaling pathway in neonatal rats. Firstly, Magnetic resonance imaging (MRI) combined with hematoxylin-eosin (H&E) staining was used to assess brain injury. Initial findings showed abnormal signals in T2-weighted imaging (T2WI) and diffusion weighted imaging (DWI). Changes also happened in the morphology of nerve cells. Subsequently, we found that BBB damage is manifested as leakage of immunoglobulin G (IgG) and destruction of BBB-related proteins and ultrastructure. Meanwhile, the levels of matrix metalloproteinase-9 (MMP-9) significantly increased at 24 h after HIBD compared to a series of time points. Additionally, immunohistochemical (IHC) staining combined with Western blot (WB) was used to verify the function of the AKT/GSK-3β/CREB signaling pathway in BBB damage after HI in neonatal rats. Results showed that less Claudin-5, ZO-1, p-AKT, p-GSK-3β and p-CREB, along with more MMP-9 protein expression were visible on the damaged side of the cerebral cortex in the HIBD group in contrast to the sham and HIBD + SC79 groups. Together, our findings demonstrated that HI in neonatal rats might upregulate the levels of MMP-9 protein and downregulate the levels of Claudin-5 and ZO-1 by inhibiting the AKT/GSK-3β/CREB pathway, thus disrupting the BBB, which in turn aggravates brain damage after HI in neonatal rats.

Neuroserpin As an Adjuvant Therapy for Hypothermia on Brain Injury in Neonatal Hypoxic-Ischemic Rats.

We aimed to assess the effects of neuroserpin and its combination with hypothermia on hypoxic-ischemic (HI) brain injury in neonatal rats. Neuroserpin is an axon-secreted serine protease inhibitor and is important for brain development, neuronal survival, and synaptic plasticity. Male Wistar-Albino rats on postnatal day 7 (P7) were randomly divided into five groups: sham group (n = 10), (HI; n = 10), hypoxic-ischemic hypothermia (HIH; n = 10), hypoxic-ischemic neuroserpin (HIN; n = 10), and hypoxic-ischemic neuroserpin-hypothermia (HINH; n = 10). The P7 rat brain's maturation is similar to a late preterm human brain at 34 to 36 weeks of gestation. HI was induced in rats on P7 as previously described. A single dose of 0.2 µM neuroserpin (HINH and HIN) or an equivalent volume of phosphate-buffered saline (sham, HIH, and HI) was administered intraventricularly by a Hamilton syringe immediately after hypoxia. In the follow-up, pups were subjected to systemic hypothermia or normothermia for 2 hours. Euthanasia was performed for histopathological evaluation on P10. Apoptosis was detected by caspase-3 activity and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and was counted in the hippocampus. In comparison to the HI group, the TUNEL-positive and caspase-3-positive neurons in the sham, HIN, HIH, and HINH groups were considerably lower (13.4 ± 1.0 vs. 1.9 ± 0.9, 6.0 ± 0.9, 5.3 ± 1.6, and 4.0 ± 1.1; p < 0.001) and (13.5 ± 1.7 vs. 1.2 ± 0.7, 9.1 ± 2.7, 4.8 ± 1.0, and 3.9 ± 1.6; p < 0.001). HIN, HIH, and HINH, compared to the sham group, showed more TUNEL-positive and caspase-3-positive neurons (6.0 ± 0.9, 5.3 ± 1.6, 4.0 ± 1.1 vs. 1.9 ± 0.9 and 9.1 ± 2.7, 4.8 ± 1.0, 3.9 ± 1.6 vs. 1.2 ± 0.7; p < 0.001). The HINH group (synergistic effect) had significantly fewer TUNEL-positive neurons and caspase-3-positive neurons than the HIN group (4.0 ± 1.1 vs. 6.0 ± 0.9 and 3.9 ± 1.6 vs. 9.1 ± 2.7; p < 0.001). Our study showed that both neuroserpin alone and as an adjuvant treatment for hypothermia may have a neuroprotective effect on brain injury. · Neuroserpin decreased brain injury.. · Neuroserpin showed a synergistic effect when used as an adjuvant treatment for hypothermia.. · The neuroprotective effect of neuroserpine was related to its antiapoptotic properties..

The Long-Term Neuroprotective Effect of the Endocannabinoid 2-AG and Modulation of the SGZ's Neurogenic Response after Neonatal Hypoxia-Ischemia.

Neonatal hypoxia-ischemia (HI) often causes hypoxic-ischemic encephalopathy (HIE), a neurological condition that can lead to overall disability in newborns. The only treatment available for affected neonates is therapeutic hypothermia; however, cooling is not always effective to prevent the deleterious effects of HI, so compounds such as cannabinoids are currently under research as new therapies. Modulating the endocannabinoid system (ECS) may reduce brain damage and/or stimulate cell proliferation at the neurogenic niches. Further, the long-term effects of cannabinoid treatment are not so clear. Here, we studied the middle- and long-term effects of 2-AG, the most abundant endocannabinoid in the perinatal period after HI in neonatal rats. At middle-term (postnatal day 14), 2-AG reduced brain injury and increased SGZ's cell proliferation and the number of neuroblasts. At post-natal day 90, the treatment with the endocannabinoid showed global and local protection, suggesting long-lasting neuroprotective effects of 2-AG after neonatal HI in rats.

Transcranial Doppler Ultrasonography detection on cerebrovascular flow for evaluating neonatal hypoxic-ischemic encephalopathy modeling.

This study aimed to investigate the feasibility of Transcranial Doppler Ultrasonography (TCD) in evaluating neonatal hypoxic-ischemic encephalopathy (NHIE) modeling through monitoring the alteration of cerebrovascular flow in neonatal hypoxic-ischemic (HI) rats. Postnatal 7-day-old Sprague Dawley (SD) rats were divided into the control group, HI group, and hypoxia (H) group. TCD was applied to assess the changes of cerebral blood vessels, cerebrovascular flow velocity, and heart rate (HR) in sagittal and coronal sections at 1, 2, 3, and 7 days after the operation. For accuracy, cerebral infarct of rats was examined by 2,3,5-Triphenyl tetrazolium chloride (TTC) staining and Nissl staining to simultaneously verify the establishment of NHIE modeling. Coronal and sagittal TCD scans revealed obvious alteration of cerebrovascular flow in main cerebral vessels. Obvious cerebrovascular back-flow was observed in anterior cerebral artery (ACA), basilar artery (BA), middle cerebral artery (MCA) of HI rats, along with accelerated cerebrovascular flows in the left internal carotid artery (ICA-L) and BA, decreased flows in right internal carotid artery (ICA-R) relative to those in the H and control groups. The alterations of cerebral blood flows in neonatal HI rats indicated successful ligation of right common carotid artery. Besides, TTC staining further validated the cerebral infarct was indeed caused due to ligation-induced insufficient blood supply. Damage to nervous tissues was also revealed by Nissl staining. Cerebral blood flow assessment by TCD in neonatal HI rats contributed to cerebrovascular abnormalities observed in a real-time and non-invasive way. The present study elicits the potentials to utilize TCD as an effective means for monitoring the progression of injury as well as NHIE modeling. The abnormal appearance of cerebral blood flow is also beneficial to the early warning and effective detection in clinical practice.

Effect of type 1 diabetes during pregnancy and lactation on neonatal hypoxia-ischemia injury and apoptotic gene expression.

Hypoxia-ischemia (HI) is one of the major disorders that causes a variety of abnormalities and infant mortality. Type 1 diabetes is one of the most common worldwide metabolic disorders, and its prevalence has become one of the public health concerns of the 21st century. This study aims to evaluate the effect of type 1 diabetes during pregnancy and lactation on vulnerability to neonatal HI in rats. Female Wistar rats (weighing 200 ± 220 g) were randomly assigned into two groups: Group 1 (rats that received 0.5mL/day of normal saline solution) and Group 2 (type 1 diabetes was induced in rats on the second day of pregnancy with a single dose of intraperitoneal injection of alloxan monohydrate [150 mg/kg]). After delivery, offspring were divided into four groups: (a) Control (Co), (b) Diabetic (DI), (c) Hypoxia-ischemia (HI), (d) HI + Diabetic (HI + DI). Seven days after HI induction, neurobehavioral tests were performed, and then cerebral edema, infarct volume, inflammatory factors, Bax-Bcl2 expression, and oxidative stress were measured. The BAX level in the DI + HI (p = 0.0355) group was significantly higher than the HI group. The Bcl-2 expression levels in the HI (p = 0.0027) and DI + HI (p < 0.0001) groups were significantly lower than the DI group. Total antioxidant capacity (TAC) levels in the DI + HI (p < 0.0001) group were significantly lower than HI and CO groups. TNF-α, CRP, and total oxidant status (TOS) levels in the DI + HI (p < 0.0001) group were significantly higher than the HI group. Infarct volume and cerebral edema in the DI + HI (p < 0.0001) group were significantly higher than the HI group. According to the results, type 1 diabetes during pregnancy and lactation increased the destructive effects of HI injury in pups. It also decreased the expression of the anti-apoptotic Bcl-2 and increased the expression BAX apoptosis factor gene in pups.

Inter-Alpha Inhibitor Proteins Modify the Microvasculature after Exposure to Hypoxia-Ischemia and Hypoxia in Neonatal Rats.

Microvasculature develops during early brain development. Hypoxia-ischemia (HI) and hypoxia (H) predispose to brain injury in neonates. Inter-alpha inhibitor proteins (IAIPs) attenuate injury to the neonatal brain after exposure to HI. However, the effects of IAIPs on the brain microvasculature after exposure to HI have not been examined in neonates. Postnatal day-7 rats were exposed to sham treatment or right carotid artery ligation and 8% oxygen for 90 min. HI comprises hypoxia (H) and ischemia to the right hemisphere (HI-right) and hypoxia to the whole body, including the left hemisphere (H-left). Human IAIPs (hIAIPs, 30 mg/kg) or placebo were injected immediately, 24 and 48 h after HI/H. The brains were analyzed 72 h after HI/H to determine the effects of hIAIPs on the microvasculature by laminin immunohistochemistry and calculation of (1) the percentage area stained by laminin, (2) cumulative microvessel length, and (3) density of tunneling nanotubes (TNTs), which are sensitive indicators of the earliest phases of neo-vascularization/collateralization. hIAIPs mainly affected the percent of the laminin-stained area after HI/H, cumulative vessel length after H but not HI, and TNT density in females but not males. hIAIPs modify the effects of HI/H on the microvasculature after brain injury in neonatal rats and exhibit sex-related differential effects. Our findings suggest that treatment with hIAIPs after exposure to H and HI in neonatal rats affects the laminin content of the vessel basal lamina and angiogenic responses in a sex-related fashion.

Hypothermia Does Not Boost the Neuroprotection Promoted by Umbilical Cord Blood Cells in a Neonatal Hypoxia-Ischemia Rat Model

Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the leading causes of death and long-term disability in the perinatal period. Currently, therapeutic hypothermia is the standard of care for this condition with modest efficacy and strict enrollment criteria. Therapy with umbilical cord blood cells (UCBC) has come forward as a strong candidate for the treatment of neonatal HIE, but no preclinical studies have yet compared the action of UCBC combined with hypothermia (HT) with the action of each therapy by itself. Thus, to evaluate the potential of each therapeutic approach, a hypoxic-ischemic brain lesion was induced in postnatal day ten rat pups; two hours later, HT was applied for 4 h; and 24, 48, and 72 h post-injury, UCBC were administered intravenously. The neonatal hypoxic-ischemic injury led to a brain lesion involving about 48% of the left hemisphere that was not improved by HT (36%) or UCBC alone (28%), but only with the combined therapies (25%; p = 0.0294). Moreover, a decrease in glial reactivity and improved functional outcomes were observed in both groups treated with UCBC. Overall, these results support UCBC as a successful therapeutic approach for HIE, even when treatment with therapeutic hypothermia is not possible.

Knockdown of interleukin-6 plays a neuroprotective role against hypoxia-ischemia in neonatal rats via inhibition of caspase 3 and Bcl-2-associated X protein signaling pathway.

This study aimed to investigate the role of interleukin-6 (IL-6) in the pathogenesis of neonatal hypoxic-ischemic encephalopathy (NHIE). Sprague-Dawley (SD) rats were used for the establishment of hypoxic-ischemic (HI) model. The Zea-Longa scoring was used to evaluate the extent of the neurological deficits. Triphenyl tetrazolium chloride (TTC) staining was used to measure the volume of infarction in the brain following HI protocol. The expression of IL-6 in the cortex and/or hippocampus at multiple time points after HI was examined by immunohistochemistry, western blotting and immunofluorescence. Moreover, small interfering RNAs (siRNA) were used to inhibit the expression of IL-6 in-vitro and in-vivo, and the concomitant expression of the Bcl-2 associated X protein (BAX) and caspase 3 was also measured. HI induced a significant brain damage, and these pathological changes were accompanied by IL-6 upregulation which was found localized in cortical neurons. The inhibition of IL-6 expression fostered neuronal and axonal growth, and a reduction in cellular apoptosis in cortical neuronal cultures, and cortex and hippocampus of neonatal rats. The expression of apoptotic markers such as BAX and caspase 3 was closely associated with IL-6. Downregulation of IL-6 could ameliorate HI-induced deficiencies by mediating the expression of caspase 3 and BAX.

The TXNIP/Trx-1/GPX4 pathway promotes ferroptosis in hippocampal neurons after hypoxia-ischemia in neonatal rats

To observe the change in ferroptosis in hippocampal neurons after hypoxia-ischemia (HI) in neonatal rats and investigate the related mechanism based on the TXNIP/Trx-1/GPX4 signaling pathway. Healthy neonatal Sprague-Dawley rats, aged 7 days, were randomly divided into three groups: sham-operation (n=30), hypoxic-ischemic brain damage (HIBD) (n=30) and siRNA (TXNIP siRNA) (n=12). The classic Rice-Vannucci method was used to establish a neonatal rat model of HIBD. At 6 hours, 24 hours, 72 hours, and 7 days after modeling, Western blot was used to measure the protein expression of GPX4 in the hippocampal tissue at the injured side; at 24 hours after modeling, laser speckle imaging combined with hematoxylin-eosin staining was used to determine whether the model was established successfully; NeuN/GPX4 and GFAP/GPX4 immunofluorescence staining combined with Western blot and other methods was used to measure the protein expression of GPX4 and the signal molecules TXNIP and Trx-1 in the hippocampal tissue at the injured side; the kits for determining the content of serum iron and tissue iron were used to measure the change in iron content; quantitative real-time PCR was used to measure the mRNA expression of TXNIP, Trx-1, and GPX4. At 6 hours, 24 hours, 72 hours, and 7 days after modeling, the HIBD group had a significantly lower protein expression level of GPX4 than the sham-operation group (P<0.05). At 24 hours after modeling, the HIBD group had a significantly lower cerebral blood flow of the injured side than the sham-operation group (P<0.05), with loose and disordered arrangement and irregular morphology of hippocampal CA1 neurons at the injured side. Compared with the sham-operation group, the HIBD group had a significantly higher number of TXNIP+ cells and significantly lower numbers of Trx-1+ cells and NeuN+GPX4+/NeuN+ cells in the hippocampal CA1 region at the injured side (P<0.05), with almost no GFAP+GPX4+ cells in the hippocampal CA1 region. Compared with the sham-operation group, the HIBD group and the siRNA group had significantly higher levels of serum iron and tissue iron in the hippocampus at the injured side (P<0.05). Compared with the HIBD group, the siRNA group had significantly lower levels of serum iron and tissue iron in the hippocampus at the injured side (P<0.05). The HIBD group and the siRNA group had significantly higher mRNA and protein expression levels of TXNIP than the sham-operation group (P<0.05), and the siRNA group had significantly lower expression levels than the HIBD group (P<0.05). The HIBD group and the siRNA group had significantly lower mRNA and protein expression levels of Trx-1 and GPX4 in the hippocampus at the injured side than the sham-operation group (P<0.05), and the siRNA group had significantly higher expression levels than the HIBD group (P<0.05). HI induces ferroptosis of hippocampal neurons in neonatal rats by activating the TXNIP/Trx-1/GPX4 pathway, thereby resulting in HIBD.

Neonatal Hypoxia-Ischemia Causes Persistent Intracortical Circuit Changes in Layer 4 of Rat Auditory Cortex.

The connection between early brain injury and subsequent development of disorders is unknown. Neonatal hypoxia-ischemia (HI) alters circuits associated with subplate neurons (SPNs). SPNs are among the first maturing cortical neurons, project to thalamorecipient layer 4 (L4), and are required for the development of thalamocortical connections. Thus, early HI might influence L4 and such influence might persist. We investigated functional circuits to L4 neurons in neonatal rat HI models of different severities (mild and moderate) shortly after injury and at adolescence. We used laser-scanning photostimulation in slices of auditory cortex during P5-10 and P18-23. Mild injuries did not initially (P6/P7) alter the convergence of excitatory inputs from L2/3, but hyperconnectivity emerged by P8-10. Inputs from L4 showed initial hypoconnectivity which resolved by P8-10. Moderate injuries resulted in initial hypoconnectivity from both layers which resolved by P8-10 and led to persistent strengthening of connections. Inhibitory inputs to L4 cells showed similar changes. Functional changes were mirrored by reduced dendritic complexity. We also observed a persistent increase in similarity of L4 circuits, suggesting that HI interferes with developmental circuit refinement and diversification. Altogether, our results show that neonatal HI injuries lead to persistent changes in intracortical connections.

Beneficial effect of treadmill exercise during pregnancy of obese mothers against neonatal hypoxia-ischemia in rats

Earlier research studies have found that maternal obesity during pregnancy contributes to neonatal hypoxia-ischemia. This study aims to examine whether treadmill workouts during pregnancy of obese mothers could attenuate neonatal hypoxia-ischemia in both male and female offspring of rat. In the present study, normal and obese pregnant Wistar rats were used in six group. Some groups underwent treadmill exercise during pregnancy. Eight days after delivery, pups underwent neonatal hypoxia-ischemia induction. Seven days after hypoxia-ischemia induction, neurobehavioral tests were performed, and then, brain tissue was taken from the skull to measure cerebral edema, infarct volume, apoptotic factors, and Interleukin 6 (IL-6). The findings of this study indicated that the neurobehavioral function and %Bcl-2 gene expression in pups that born from obese mothers who exercise during pregnancy was significantly better than pups of obese mothers without exercise during pregnancy. Infarct volume, edema, BAX and IL-6 levels in pups that born from obese mothers who exercise during pregnancy was significantly lower than pups of obese mothers without exercise during pregnancy. It seems that maternal treadmill exercise during pregnancy of obese mothers has beneficial effects against hypoxia-ischemia injury in rat pups. • Maternal obesity during pregnancy contributes to neonatal hypoxia-ischemia (HI). • In obese dam treadmill exercise during pregnancy reduced neonatal HI damage. • Treadmill exercise during pregnancy reduced apoptotic factors in pups brain.

NAC and Vitamin D Restore CNS Glutathione in Endotoxin-Sensitized Neonatal Hypoxic-Ischemic Rats.

Therapeutic hypothermia does not improve outcomes in neonatal hypoxia ischemia (HI) complicated by perinatal infection, due to well-described, pre-existing oxidative stress and neuroinflammation that shorten the therapeutic window. For effective neuroprotection post-injury, we must first define and then target CNS metabolomic changes immediately after endotoxin-sensitized HI (LPS-HI). We hypothesized that LPS-HI would acutely deplete reduced glutathione (GSH), indicating overwhelming oxidative stress in spite of hypothermia treatment in neonatal rats. Post-natal day 7 rats were randomized to sham ligation, or severe LPS-HI (0.5 mg/kg 4 h before right carotid artery ligation, 90 min 8% O2), followed by hypothermia alone or with N-acetylcysteine (25 mg/kg) and vitamin D (1,25(OH)2D3, 0.05 μg/kg) (NVD). We quantified in vivo CNS metabolites by serial 7T MR Spectroscopy before, immediately after LPS-HI, and after treatment, along with terminal plasma drug concentrations. GSH was significantly decreased in all LPS-HI rats compared with baseline and sham controls. Two hours of hypothermia alone did not improve GSH and allowed glutamate + glutamine (GLX) to increase. Within 1 h of administration, NVD increased GSH close to baseline and suppressed GLX. The combination of NVD with hypothermia rapidly improved cellular redox status after LPS-HI, potentially inhibiting important secondary injury cascades and allowing more time for hypothermic neuroprotection.