Cortex Of Neonatal Rats Research Articles

3-Hydroxy-3-Methylglutaric Acid Disrupts Brain Bioenergetics, Redox Homeostasis, and Mitochondrial Dynamics and Affects Neurodevelopment in Neonatal Wistar Rats.

3-Hydroxy-3-methylglutaric acidemia (HMGA) is a neurometabolic inherited disorder characterized by the predominant accumulation of 3-hydroxy-3-methylglutaric acid (HMG) in the brain and biological fluids of patients. Symptoms often appear in the first year of life and include mainly neurological manifestations. The neuropathophysiology is not fully elucidated, so we investigated the effects of intracerebroventricular administration of HMG on redox and bioenergetic homeostasis in the cerebral cortex and striatum of neonatal rats. Neurodevelopment parameters were also evaluated. HMG decreased the activity of glutathione reductase (GR) and increased catalase (CAT) in the cerebral cortex. In the striatum, HMG reduced the activities of superoxide dismutase, glutathione peroxidase, CAT, GR, glutathione S-transferase, and glucose-6-phosphate dehydrogenase. Regarding bioenergetics, HMG decreased the activities of succinate dehydrogenase and respiratory chain complexes II-III and IV in the cortex. HMG also decreased the activities of citrate synthase and succinate dehydrogenase, as well as complex IV in the striatum. HMG further increased DRP1 levels in the cortex, indicating mitochondrial fission. Finally, we found that the HMG-injected animals showed impaired performance in all sensorimotor tests examined. Our findings provide evidence that HMG causes oxidative stress, bioenergetic dysfunction, and neurodevelopmental changes in neonatal rats, which may explain the neuropathophysiology of HMGA.

Exposure to lipid mixture induces intracellular lipid droplet formation and impairs mitochondrial functions in astrocytes

Astrocytes, the predominant glial cells in the central nervous system (CNS), play diverse roles including metabolic support for neurons, provision of neurotrophic factors, facilitation of synaptic neurotransmitter uptake, regulation of ion balance, and involvement in synaptic formation. The accumulation of lipids has been noted in various neurological conditions, yet the response of astrocytes to lipid-rich environments remains unclear. In this study, primary astrocytes isolated from the neonatal rat cortex were exposed to a lipid mixture (LM) comprising cholesterol and various fatty acids to explore their reaction. Our results showed that astrocyte viability remained unchanged following 24 h of 5% or 10% LM treatment. However, exposure to LM for 96 h resulted in reduced cell viability. In addition, LM treatment led to the accumulation of lipid droplets (LDs) in astrocytes, with LD size increasing over prolonged exposure periods. Following 24 h of LM treatment and then 48 h in fresh medium, a significant reduction in intracellular LD size was observed in cultures treated with 5% LM, while no change occurred in cultures exposed to 10% LM. Yet, exposure to 10% LM for 24 h significantly increased the expression of the cholesterol efflux regulatory protein/ATP-binding cassette transporter (ABCA1) gene, responsible for intracellular cholesterol efflux, resulting in reduced cholesterol content within astrocytes. Moreover, LM exposure led to decreased mitochondrial membrane potential (MMP) and increased levels of mature apoptosis-inducing factor (AIF). The smaller LDs were observed to co-localize with microtubule-associated protein 1A/1 B light chain 3 B (LC3) and lysosomal-associated membrane protein-1 (LAMP-1) in LM-treated astrocytes, coinciding with lysosomal acidification. These results indicate that the continuous buildup of LDs in astrocytes residing in lipid-enriched environments may be attributed to disruptions caused by LM in mitochondrial and lysosomal functions. Such disruptions could potentially impede the supportive role of astrocytes in neuronal function.

Jisuikang formula promotes spinal cord injury repair in rats by activating the YAP/PKM2 signaling axis in astrocytes

To investigate the effect of Jisuikang formula-medicated serum for promoting spinal cord injury (SCI) repair in rats and explore the possible mechanism. Thirty adult SD rats were randomized into sham-operated group, SCI (induced using a modified Allen method) model group, and Jisuikang formula-medicated serum treatment group. After the operations, the rats were treated with normal saline or Jisuikang by gavage on a daily basis for 14 days, and the changes in hindlimb motor function of the rats was assessed with Basso-Beattie-Bresnahan (BBB) scores and inclined-plate test. The injured spinal cord tissues were sampled from the SCI rat models for single-cell RNA sequencing, and bioinformatics analysis was performed to identify the target genes of Jisuikang, spinal cord injury and glycolysis. In the cell experiment, cultured astrocytes from neonatal SD rat cortex were treated with SOX2 alone or in combination with Jisuikang-medicated serum for 21 days, and the protein expressions of PKM2, p-PKM2 and YAP and colocalization of PKM2 and YAP in the cells were analyzed with Western blotting and immunofluorescence staining, respectively. The SCI rats with Jisuikang treatment showed significantly improved BBB scores and performance in inclined-plate test. At the injury site, high PKM2 expression was detected in various cell types. Bioinformatic analysis identified the HIPPO-YAP signaling pathway as the target pathway of Jisuikang. In cultured astrocytes, SOX2 combined with the mediated serum, as compared with SOX2 alone, significantly increased PKM2, p-PKM2 and YAP expressions and entry of phosphorylated PKM2 into the nucleus, and promoted PKM2 and YAP co-localization in the cells. Jisuikang formula accelerates SCI repair in rats possibly by promoting aerobic glycolysis of the astrocytes via activating the PKM2/YAP axis to induce reprogramming of the astrocytes into neurons.

Cortical and Striatal Astrocytes of Neonatal Rats Display Distinct Molecular and Pharmacological Characteristics of Dopamine Uptake.

Astrocytes are crucial in the regulation of neurotransmitter homeostasis, and while their involvement in the dopamine (DA) tripartite synapse is acknowledged, it necessitates a more comprehensive investigation. In the present study, experiments were conducted on primary astrocyte cultures from the striatum and cortex of neonatal rats. The pharmacological intricacies of DA uptake, including dependence on time, temperature, and concentration, were investigated using radiolabelled [3H]-DA. The mRNA expression of transporters DAT, NET, PMAT, and OCTs was evaluated by qPCR. Notably, astrocytes from both brain regions exhibited prominent mRNA expression of NET and PMAT, with comparatively lower expression of DAT and OCTs. The inhibition of DA uptake by the DAT inhibitor, GBR12909, and NET inhibitors, desipramine and nortriptyline, impeded DA uptake in striatal astrocytes more than in cortical astrocytes. The mRNA expression of NET and PMAT was significantly upregulated in cortical astrocytes in response to the DA receptor agonist apomorphine, while only the mRNA expression of NET exhibited changes in striatal astrocytes. Haloperidol, a DA receptor antagonist, and L-DOPA, a DA precursor, did not induce significant alterations in transporter mRNA expression. These findings underscore the intricate and region-specific mechanisms governing DA uptake in astrocytes, emphasizing the need for continued exploration to unravel the nuanced dynamics of astrocytic involvement in the DA tripartite synapse.

Lipopolysaccharide impairs neurodevelopment and induces changes in astroglial reactivity, antioxidant defenses and bioenergetics in the cerebral cortex of neonatal rats.

Neonates have an immature immune system, which increases their vulnerability to infectious agents and inflammatory insults. The administration of the immunostimulatory agent lipopolysaccharide (LPS) has been shown to induce the expression of pro-inflammatory cytokines and cause behavior alterations in rodents at different ages. However, the effects of LPS administration during the neonatal period and its consequences during immune system maturation remain to be elucidated. We showed here that a single intraperitoneal administration of LPS in rats on postnatal day (PND) 7 caused early and variable alterations in TNF-α, S100B and GFAP levels in the cerebral cortex, CSF and serum of the animals, indicating long-term induction of neuroinflammation and astroglial reactivity. However, on PND 21, only GFAP levels were increased by LPS. Additionally, LPS induced oxidative stress and altered energy metabolism enzymes in the cerebral cortex on PND 21, and caused neurodevelopment impairment over time. These data suggest that neuroinflammation induction during the neonatal period induces glial reactivity, oxidative stress and bioenergetic disruption that may lead to neurodevelopment impairment and cognitive deficit in adult life.

Pathophysiological aspects of neonatal anoxia and temporal expression of S100β in different brain regions.

The aim of this study was to investigate the temporal variations of S100β in the hippocampus, cerebellum and cerebral cortex of neonatal rats (Wistar strain) under anoxic conditions. Real-time PCR and western blotting techniques were used for gene expression and protein analysis. Animals were divided into two groups, a control group and an anoxic group, and further separated at different time points for analysis. After anoxia, S100β gene expression showed a significant peak in the hippocampus and cerebellum after 2 h, followed by a decline compared to the control group at other time points. The increased gene expression in these regions was also accompanied by an increase in S100β protein levels in the anoxia group, observable 4 h after injury. In contrast, S100β mRNA content in the cerebral cortex never exceeded control values at any time point. Similarly, the protein content of S100β in the cerebral cortex did not show statistically significant differences compared to control animals at any assessment time point. These results suggest that the production profile of S100β differs by brain region and developmental stage. The observed differences in vulnerability between the hippocampus, cerebellum and cerebral cortex may be attributed to their distinct developmental periods. The hippocampus and cerebellum, which develop earlier than the cerebral cortex, showed more pronounced effects in response to anoxia, which is supported by the gene expression and protein content in this study. This result reveals the brain region-dependent nature of S100β as a biomarker of brain injury.

Nimodipine exerts a protective effect against spreading depolarization and neuroinflammation

Hypothesis, objective: Voltage gated Ca2+ channels (VGCCs) are widely expressed in the central nervous system and implicated in neuronal injury and microglial activation. Nimodipine, a potent cerebrovascular dilator and VGCC antagonist is found to be effective against the vasoconstriction caused by spreading depolarization (SD) in preclinical studies of ischemia. However, it is still unclear whether the beneficial effect of nimodipine is achieved by the improvement of perfusion or a direct action on neuronal or glial cells. Our aim was to explore the direct action of nimodipine on the nervous tissue and microglia. Methods: Brain slices prepared from C57BL/6 mice (n=16) were perfused with artificial cerebrospinal fluid (aCSF). After 30 min nimodipine (nimo; 10 μM) incubation, low glucose aCSF (5 mM) and transient anoxia (1 min) were applied to mimic ischemia and to elicit SD. Intrinsic optical signal imaging was used to analyze SD features, TTC staining was applied to assess tissue injury. Primary mixed and pure microglia cultures prepared from the cortex of neonatal SPRD rats were treated with lipopolysaccharide (LPS;20 ng/ml) and nimodipine (5,10,20 μM), alone or in combination for 24 h. Microglial activation was evaluated by Iba1 immunolabeling (degree of arborization expressed by a transformation index (TI)), Western blot analysis and the visualization of phagocytosis with fluorescent microbeads. Results: In brain slices, nimodipine reduced the focal area of SD (2.37±0.94 vs 3.38±0.88%, nimo vs control), the total cortical area affected by SD (17.12±8.63 vs 39.88±22.42%, nimo vs control) and the propagation velocity of SD (0.19±0.79 vs 1.59±2.29mm/min, nimo vs control). Nimodipine decreased tissue injury, viability expressed as the number of TTC-stained particles (4.48±1.45 vs 3.52±1.52 particle/1000 μm2, nimo vs control). Nimodipine reduced microglial activation in LPS-treated cultures reflected by more ramified cell morphology (3.1±0.91 vs 1.89±0.41 TI, LPS+nimo vs LPS), decreased Iba1 intensity in Western blot analysis (108.3±20.1 and 77.2±8.49 vs 213.4±29.7 integrated optical density, LPS+nimo 10 and 20 μM vs LPS) and attenuated phagocytic activity (6±10 vs 22±20 bead/cell LPS+nimo vs LPS). Conclusion: Nimodipine was protective against SD and successfully reduced neuroinflammation by decreasing microglial activation. Neuroprotection was achieved independent of nimodipine’s vascular action. Our data suggest that nimodipine may be applicable against neuroinflammation and ischemia, in which neurodegeneration is believed to be linked to neurotoxic microglial activation. NKFIH K134377 and K134334, H2020 No. 739593, NAP3.0, TKP2021-EGA-28 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sulfite Impairs Bioenergetics and Redox Status in Neonatal Rat Brain: Insights into the Early Neuropathophysiology of Isolated Sulfite Oxidase and Molybdenum Cofactor Deficiencies.

Isolated sulfite oxidase (ISOD) and molybdenum cofactor (MoCD) deficiencies are genetic diseases biochemically characterized by the toxic accumulation of sulfite in the tissues of patients, including the brain. Neurological dysfunction and brain abnormalities are commonly observed soon after birth, and some patients also have neuropathological alterations in the prenatal period (in utero). Thus, we investigated the effects of sulfite on redox and mitochondrial homeostasis, as well as signaling proteins in the cerebral cortex of rat pups. One-day-old Wistar rats received an intracerebroventricular administration of sulfite (0.5µmol/g) or vehicle and were euthanized 30min after injection. Sulfite administration decreased glutathione levels and glutathione S-transferase activity, and increased heme oxygenase-1 content in vivo in the cerebral cortex. Sulfite also reduced the activities of succinate dehydrogenase, creatine kinase, and respiratory chain complexes II and II-III. Furthermore, sulfite increased the cortical content of ERK1/2 and p38. These findings suggest that redox imbalance and bioenergetic impairment induced by sulfite in the brain are pathomechanisms that may contribute to the neuropathology of newborns with ISOD and MoCD. Sulfite disturbs antioxidant defenses, bioenergetics, and signaling pathways in the cerebral cortex of neonatal rats. CII: complex II; CII-III: complex II-III; CK: creatine kinase; GST: glutathione S-transferase; HO-1: heme oxygenase-1; SDH: succinate dehydrogenase; SO32-: sulfite.

Role of hyperbaric oxygen therapy in PDGF-BB-mediated astrogliosis in traumatic brain injury rats associated with ERK1/2 signaling pathway inhibition

BackgroundHyperbaric oxygen (HBO) plays positive roles in the therapy of traumatic brain injury (TBI); however, the mechanism underlying its effects on TBI is largely unknown. The study aims to elucidate the molecular mechanism implicated with the interaction between platelet-derived growth factor-BB (PDGF-BB) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway, which may play critical roles during HBO treatment both in the astrocyte scratching model in vitro and rat TBI model in vivo.MethodsChanges in neurological function and wound healing were evaluated using the neurological severity scores (NSS) scale, immunohistochemistry, western blotting, and qRT-PCR, respectively.ResultsThe results showed that PDGF-BBi (PDGB interfered with small RNA) dramatically improves neuronal viability in vitro when transfected into the scratched astrocytes derived from the cerebral cortex of neonatal rats. Moreover, in vivo experiments revealed that HBO therapy substantially elevated the NSS scores and simultaneously reduced the mortality in TBI rats, as indicated by the NSS scales. Notably, HBO therapy was found to possess the ability to inhibit glial cell proliferation, promote the regeneration of neurons and synapses, and ultimately facilitate the wound healing, as revealed by immunohistochemistry and glial scar formation found in TBI rats. Importantly, HBO markedly decreased the expression levels of PDGF-BB and ERK1/2. It can clearly be seen that downregulated PDGF-BB and ERK1/2 levels were corresponding with the status of significant amelioration of the therapeutic effect of HBO. Conversely, the upregulation of PDGF-BB and ERK1/2 levels was in line with the opposite effect.ConclusionIt has been concluded that HBO therapy may play its active role in TBI treatment dependent on astrogliosis inhibition, which may be achieved by downregulating the ERK1/2 signaling pathway mediated by PDGF-BB.

Sevoflurane Exposure of Clinical Doses in Pregnant Rats Induces Vcan Changes without Significant Neural Apoptosis in the Offspring

Background and Objectives: Sevoflurane is a commonly used inhalational anaesthetic in clinics. Prolonged exposure to sevoflurane can induce significant changes in lipid metabolism and neuronal damage in the developing brain. However, the effect of exposure of pregnant rats to clinical doses of sevoflurane remains unclear. Materials and Methods: Twenty-eight pregnant rats were randomly and equally divided into sevoflurane exposure (S) group, control (C) and a blank group at gestational day (G) 18; Rats in S group received 2% sevoflurane with 98% oxygen for 6 h in an anesthetizing chamber, while C group received 100% oxygen at an identical flow rate for 6 h in an identical chamber. Partial least squares discriminant analysis (PLS-DA), ultra performance liquid chromatography/time-of-flight mass spectrometry(UPLC/TOF-MS) and MetaboAnalyst were used to analysis acquire metabolomics profiles, and immunohistochemical changes of neuronalapoptosis in hippocampus and cortex of neonatal rats were also analyzed. Results: This study aimed to explore lipidomics and transcriptomics changes related to 2% sevoflurane exposure for 6 h in the developing brains of newborn offspring rats. Ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOF-MS) and RNA sequencing (RNA-seq) analyses were used to acquire metabolomics and transcriptomics profiles. We used RNA-seq to analyse the expression of the coding and non-coding transcripts in neural cells of the cerebral cortex. No significant differences in arterial oxygen tension (PaO2), arterial carbon dioxide tension (PaCO2), or arterial blood gas were found between the groups. The relative standard deviation (RSD) of retention times was <1.53%, and the RSDs of peak areas ranged from 2.13% to 8.51%. Base peak chromatogram (BPC) profiles showed no differences between the groups. We evaluated the partial least square-discriminant analysis (PLS-DA) model. In negative ion mode, R2X was over 70%, R2Y was over 93%, and Q2 (cum) was over 80%. Cell apoptosis was not remarkably enhanced by TUNEL and haematoxylin and eosin (HE) staining in the sevoflurane-exposed group compared to the control group (p > 0.05). Glycerophospholipid (GP) and sphingolipid metabolism disturbances might adversely influence neurodevelopment in offspring. The expression of mRNAs (Vcan gene, related to neuronal development, function and repair) of the sevoflurane group was significantly increased in the differential genes by qRT-PCR verification. Conclusions: GP and sphingolipid metabolism homeostasis may be potential therapeutic approaches against inhalational anaesthetic-induced neurodegenerative disorders. Meanwhile, sevoflurane-induced Vcan changes indicated some lipidomic and transcriptomic changes, even if neural cell apoptosis was not significantly changed in the usual clinical dose of sevoflurane exposure.

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.

AQP4 knockout promotes neurite outgrowth via upregulating GAP43 expression in infant rats with hypoxic-ischemic brain injury.

Neonatal hypoxic-ischemic encephalopathy (NHIE) induces severe cerebral damage and neurological dysfunction, with seldom effective therapy. Aquaporin-4 (AQP4)isinvolvedin aggravating brain damage induced by NHIE. This study aimed to investigate the role of AQP4 underlying the pathogenesis of NHIE. Neonatal Sprague-Dawley rats were used to establish neonatal hypoxic-ischemic (HI) models, and the expression of AQP4 in the cortex, hippocampus, and lung tissues was detected by real-time quantitative polymerase chain reaction as well as Western blot. Primary cortical neurons were cultured for the oxygen-glucose deprivation (OGD) model, and siRNA was used to silence the expression of AQP4. Immunostaining of Tuj1 was performed to observe the axonal growth. CRISPER/Cas9 technology was used to knock out AQP4. The results demonstrated thatAQP4 was upregulated in the cortex, hippocampus, and lung tissues in neonatal rats with HI andOGD neurons. Besides, silencing AQP4 promoted axonal growth of OGD neurons, and AQP4 knockout notably improved long-term neurobehavioral impairment. Furthermore, GAP43 was found closely correlated with AQP4 via GeneMANIA prediction. Significant downregulation of GAP43 was induced in OGD neurons, while AQP4 knockout markedly upregulated its expression in rats. This indicated that the depletion of AQP4 may enhance axonal regeneration and promote the long-term neurobehavioral recovery associated with the upregulation of GAP43 expression.

High Mobility Group A1 Regulates Transcription Levels of Oligodendrocyte Marker Genes in Cultured Oligodendrocyte Precursor Cells.

Oligodendrocyte precursor cells (OPCs) serve as progenitor cells of terminally differentiated oligodendrocytes. Past studies have confirmed the importance of epigenetic system in OPC differentiation to oligodendrocytes. High mobility group A1 (HMGA1) is a small non-histone nuclear protein that binds DNA and modifies the chromatin conformational state. However, it is still completely unknown about the roles of HMGA1 in the process of OPC differentiation. In this study, we prepared primary OPC cultures from the neonatal rat cortex and examined whether the loss- and gain-of-function of HMGA1 would change the mRNA levels of oligodendrocyte markers, such as Cnp, Mbp, Myrf and Plp during the process of OPC differentiation. In our system, the mRNA levels of Cnp, Mbp, Myrf and Plp increased depending on the oligodendrocyte maturation step, but the level of Hmga1 mRNA decreased. When HMGA1 was knocked down by a siRNA approach, the mRNA levels of Cnp, Mbp, Myrf and Plp were smaller in OPCs with Hmga1 siRNA compared to the ones in the control OPCs. On the contrary, when HMGA1 expression was increased by transfection of the Hmga1 plasmid, the mRNA levels of Cnp, Mbp, Myrf and Plp were slightly larger compared to the ones in the control OPCs. These data may suggest that HMGA1 participates in the process of OPC differentiation by regulating the mRNA expression level of myelin-related genes.

EGF Downregulates Presynaptic Maturation and Suppresses Synapse Formation In Vitro and In Vivo.

Neuronal differentiation, maturation, and synapse formation are regulated by various growth factors. Here we show that epidermal growth factor (EGF) negatively regulates presynaptic maturation and synapse formation. In cortical neurons, EGF maintained axon elongation and reduced the sizes of growth cones in culture. Furthermore, EGF decreased the levels of presynaptic molecules and number of presynaptic puncta, suggesting that EGF inhibits neuronal maturation. The reduction of synaptic sites is confirmed by the decreased frequencies of miniature EPSCs. In vivo analysis revealed that while peripherally administrated EGF decreased the levels of presynaptic molecules and numbers of synaptophysin-positive puncta in the prefrontal cortices of neonatal rats, EGF receptor inhibitors upregulated these indexes, suggesting that endogenous EGF receptor ligands suppress presynaptic maturation. Electron microscopy further revealed that EGF decreased the numbers, but not the sizes, of synaptic structures in vivo. These findings suggest that endogenous EGF and/or other EGF receptor ligands negatively modulates presynaptic maturation and synapse formation.

Autism-Related Transcription Factors Underlying the Sex-Specific Effects of Prenatal Bisphenol A Exposure on Transcriptome-Interactome Profiles in the Offspring Prefrontal Cortex.

Bisphenol A (BPA) is an environmental risk factor for autism spectrum disorder (ASD). BPA exposure dysregulates ASD-related genes in the hippocampus and neurological functions of offspring. However, whether prenatal BPA exposure has an impact on genes in the prefrontal cortex, another brain region highly implicated in ASD, and through what mechanisms have not been investigated. Here, we demonstrated that prenatal BPA exposure disrupts the transcriptome–interactome profiles of the prefrontal cortex of neonatal rats. Interestingly, the list of BPA-responsive genes was significantly enriched with known ASD candidate genes, as well as genes that were dysregulated in the postmortem brain tissues of ASD cases from multiple independent studies. Moreover, several differentially expressed genes in the offspring’s prefrontal cortex were the targets of ASD-related transcription factors, including AR, ESR1, and RORA. The hypergeometric distribution analysis revealed that BPA may regulate the expression of such genes through these transcription factors in a sex-dependent manner. The molecular docking analysis of BPA and ASD-related transcription factors revealed novel potential targets of BPA, including RORA, SOX5, TCF4, and YY1. Our findings indicated that prenatal BPA exposure disrupts ASD-related genes in the offspring’s prefrontal cortex and may increase the risk of ASD through sex-dependent molecular mechanisms, which should be investigated further.

The Increased Channel Activity of N-Methyl-D-Aspartate Receptors at Extrasynaptic Sites in the Anterior Cingulate Cortex of Neonatal Rats Following Prolonged Ketamine Exposure.

BackgroundKetamine is a dissociative anesthetic, commonly used for analgesia and anesthesia in a variety of pediatric procedures. It acts as a non-competitive antagonist to block ion channels of the N-methyl-D-aspartate receptors (NMDARs). Our previous study showed that repeated ketamine exposure developed a compensatory increase in NMDAR-mediated currents in neurons of the anterior cingulate cortex (ACC) of neonatal rats, and this increase was largely mediated by the GluN2B subunit-containing receptors, a predominant type of NMDARs during embryonic and early development of the brain. These data provide the molecular evidence to support that immature neurons are highly vulnerable to the development of apoptotic cell death after prolonged ketamine exposure.MethodsUsing whole-cell patch-clamp electrophysiology in an in vitro preparation of rat forebrain slices containing the ACC, the present study aimed at further determining whether GluN2B-containing NMDARs at extrasynaptic sites of immature neurons were the major target of ketamine for developing a compensatory increase in NMDAR-mediated synaptic transmission.ResultsOur major findings were that GluN2B subunits played a significant role in mediating ketamine-induced blockade of NMDAR-mediated currents in neonatal neurons and GluN2B-containing NMDARs expressed at extrasynaptic sites in neonatal neurons were the major player in compensatory enhancement of NMDAR-mediated currents after repeated ketamine exposure.ConclusionThese results provide new evidence to strongly indicate that GluN2B-containing NMDARs at extrasynaptic sites are the key molecule contributing to the high vulnerability of the neonatal brain to ketamine-induced neurotoxic effects.

Stem cell restores thalamocortical plasticity to rescue cognitive deficit in neonatal intraventricular hemorrhage

Severe neonatal intraventricular hemorrhage (IVH) patients incur long-term neurologic deficits such as cognitive disabilities. Recently, the intraventricular transplantation of allogeneic human umbilical cord blood-derived mesenchymal stem cells (MSCs) has drawn attention as a therapeutic potential to treat severe IVH. However, its pathological synaptic mechanism is still elusive. We here demonstrated that the integration of the somatosensory input was significantly distorted by suppressing feed-forward inhibition (FFI) at the thalamocortical (TC) inputs in the barrel cortices of neonatal rats with IVH by using BOLD-fMRI signal and brain slice patch-clamp technique. This is induced by the suppression of Hebbian plasticity via an increase in tumor necrosis factor-α expression during the critical period, which can be effectively reversed by the transplantation of MSCs. Furthermore, we showed that MSC transplantation successfully rescued IVH-induced learning deficits in the sensory-guided decision-making in correlation with TC FFI in the layer 4 barrel cortex.

High-mobility group box-1 and inter-alpha inhibitor proteins: In vitro binding and co-localization in cerebral cortex after hypoxic-ischemic injury.

The high-mobility group box-1 (HMGB1) protein is a transcription-regulating protein located in the nucleus. However, it serves as a damage-associated molecular pattern protein that activates immune cells and stimulates inflammatory cytokines to accentuate neuroinflammation after release from damaged cells. In contrast, Inter-alpha Inhibitor Proteins (IAIPs) are proteins with immunomodulatory effects including inhibition of pro-inflammatory cytokines. We have demonstrated that IAIPs exhibit neuroprotective properties in neonatal rats exposed to hypoxic-ischemic (HI) brain injury. In addition, previous studies have suggested that the light chain of IAIPs, bikunin, may exert its anti-inflammatory effects by inhibiting HMGB1 in a variety of different injury models in adult subjects. The objectives of the current study were to confirm whether HMGB1 is a target of IAIPs by investigating the potential binding characteristics of HMGB1 and IAIPs in vitro, and co-localization in vivo in cerebral cortices after exposure to HI injury. Solid-phase binding assays and surface plasmon resonance (SPR) were used to determine the physical binding characteristics between IAIPs and HMGB1. Cellular localizations of IAIPs-HMGB1 in neonatal rat cortex were visualized by double labeling with anti-IAIPs and anti-HMGB1 antibodies. Solid-phase binding and SPR demonstrated specific binding between IAIPs and HMGB1in vitro. Cortical cytoplasmic and nuclear co-localization of IAIPs and HMGB1 were detected by immunofluorescent staining in control and rats immediately and 3hours after HI. In conclusion, HMGB1 and IAIPs exhibit direct binding in vitro and co-localization in vivo in neonatal rats exposed to HI brain injury suggesting HMGB1 could be a target of IAIPs.

Characterization of Glycosaminoglycan Disaccharide Composition in Astrocyte Primary Cultures and the Cortex of Neonatal Rats.

Astrocytes are major producers of the extracellular matrix (ECM), which is involved in the plasticity of the developing brain. In utero alcohol exposure alters neuronal plasticity. Glycosaminoglycans (GAGs) are a family of polysaccharides present in the extracellular space; chondroitin sulfate (CS)- and heparan sulfate (HS)-GAGs are covalently bound to core proteins to form proteoglycans (PGs). Hyaluronic acid (HA)-GAGs are not bound to core proteins. In this study we investigated the contribution of astrocytes to CS-, HS-, and HA-GAG production by comparing the makeup of these GAGs in cortical astrocyte cultures and the neonatal rat cortex. We also explored alterations induced by ethanol in GAG and core protein levels in astrocytes. Finally, we investigated the relative expression in astrocytes of CS-PGs of the lectican family of proteins, major components of the brain ECM, in vivo using translating ribosome affinity purification (TRAP) (in Aldh1l1-EGFP-Rpl10a mice. Cortical astrocytes produce low levels of HA and show low expression of genes involved in HA biosynthesis compared to the whole developing cortex. Astrocytes have high levels of chondroitin-0-sulfate (C0S)-GAGs (possibly because of a higher sulfatase enzyme expression) and HS-GAGs. Ethanol upregulates C4S-GAGs as well as brain-specific lecticans neurocan and brevican, which are highly enriched in astrocytes of the developing cortex in vivo. These results begin to elucidate the role of astrocytes in the biosynthesis of CS- HS- and HA-GAGs, and suggest that ethanol-induced alterations of neuronal development may be in part mediated by increased astrocyte GAG levels and neurocan and brevican expression.

Impact of mesenchymal stem cells derived conditioned media on neural progenitor cells

Neurodegenerative diseases are common problem for companion animals. Due to the limited ability of injured axons to regenerate, innovative therapies combined with rehabilitation have been applied and evaluated. Among them, stem cells and their conditioned media implantation, which can ameliorate damaged tissue has been suggested as a promising treatment strategy. The main goal of our study was to characterize mesenchymal stem cells (MSC) derived from canine adipose tissue (AT-MSC) and umbilical cord (UC-MSC) and analyse effect of their conditioned media (CM) on neurite outgrowth of neural progenitor cells isolated from the brain cortex of neonatal rats. MSC from both sources showed high osteogenic and chondrogenic potential and expression of CD90 and CD29. Furthermore, both UC-MSCCM and AT-MSCCM stimulated neurite growth. Interestingly, this effect was more pronounced with UC-MSCCM when compared to AT-MSCCM in vitro, which may be related to the different content of neurotrophic factors included in the CM.