OBJECTIVES: To investigate the effects of hyperoxia on the expression of N-methyl-D-aspartate receptor 1 (NMDAR1) and its synapse-associated molecules, including cannabinoid receptor 1 (CB1R), postsynaptic density 95 (PSD95), and synapsin (SYN), in the hippocampus of neonatal rats. METHODS: One-day-old Sprague-Dawley neonatal rats were randomly divided into a hyperoxia group and a control group (n=8 per group). The hyperoxia group was exposed to 80% ± 5% oxygen continuously, while the control group was exposed to room air, for 7 days. At 1, 3, and 7 days after hyperoxia exposure, hematoxylin and eosin (HE) staining was used to observe histopathological changes in the brain. The expression levels of NMDAR1, CB1R, PSD95, and SYN proteins and mRNAs in the hippocampus were detected by immunohistochemistry, Western blotting, and quantitative real-time PCR. RESULTS: After 7 days of hyperoxia exposure, the hyperoxia group showed decreased neuronal density and disordered arrangement in brain tissue. Compared with the control group, after 1 day of hyperoxia exposure, CB1R mRNA and both NMDAR1 and CB1R protein expression in the hyperoxia group were significantly downregulated, while SYN protein expression was significantly upregulated (P<0.05). After 3 days, mRNA expression of NMDAR1, CB1R, and SYN was significantly decreased (P<0.05); NMDAR1 and CB1R protein expression was significantly downregulated (P<0.05), while PSD95 and SYN protein expression was significantly upregulated (P<0.05). After 7 days of hyperoxia, the protein expression of NMDAR1 and CB1R was significantly upregulated (P<0.05). CONCLUSIONS Continuous hyperoxia exposure induces time-dependent changes in the expression levels of NMDAR1 and its synapse-associated molecules in the hippocampus of neonatal rats.
Animals ; Receptors, N-Methyl-D-Aspartate/genetics* ; Rats, Sprague-Dawley ; Hippocampus/pathology* ; Rats ; Animals, Newborn ; Receptor, Cannabinoid, CB1/genetics* ; Hyperoxia/metabolism* ; Disks Large Homolog 4 Protein/genetics* ; Synapsins/genetics* ; Synapses ; Male ; Female ; RNA, Messenger/analysis*

OBJECTIVES: To investigate whether mesenchymal stem cell-derived exosomes (MSC-Exo) alleviate white matter damage (WMD) in neonatal rats by targeting the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3). METHODS: Three-day-old Sprague-Dawley rats were randomly assigned to four groups: Sham, hypoxia-ischemia (HI), MSC-Exo, and MCC950 (NLRP3 inhibitor) (n=24 per group). The WMD model was established by unilateral common carotid artery ligation combined with hypoxia. Exosomes (1×10⁸ particles/μL) were transplanted into the lateral ventricle using stereotaxic guidance. Fourteen days after modeling, hematoxylin-eosin staining was used to observe pathological changes in brain tissue, and transmission electron microscopy was used to assess myelinated axons. Western blotting was performed to detect the expression of myelin basic protein (MBP), NLRP3, caspase-1, and interleukin-1β (IL-1β). Immunohistochemistry was used to measure NLRP3, caspase-1, and IL-1β expression. Twenty-eight days post-modeling, behavioral changes were evaluated using the Morris water maze. RESULTS: In the HI group, marked inflammatory cell infiltration, extensive vacuolation, and decreased numbers of myelinated axons were observed compared to the Sham group. The MSC-Exo group showed reduced inflammatory infiltration, fewer vacuoles, and increased myelinated axons compared to the HI group, while the MCC950 group showed nearly normal cell morphology. Compared to the Sham group, the HI group exhibited decreased MBP expression, fewer platform crossings, shorter time in the target quadrant, increased expression of NLRP3, caspase-1, and IL-1β, and longer escape latency (all P<0.05). Compared to the HI group, the MSC-Exo and MCC950 groups showed increased MBP expression, more platform crossings, longer target quadrant stay, and reduced NLRP3, caspase-1, and IL-1β expression, as well as shorter escape latency (all P<0.05). CONCLUSIONS MSC-Exo may attenuate white matter damage in neonatal rats by targeting the NLRP3 inflammasome and promoting oligodendrocyte maturation.
Animals ; NLR Family, Pyrin Domain-Containing 3 Protein/antagonists & inhibitors* ; Rats, Sprague-Dawley ; White Matter/pathology* ; Inflammasomes/physiology* ; Rats ; Animals, Newborn ; Mesenchymal Stem Cells ; Interleukin-1beta/analysis* ; Male ; Caspase 1/analysis* ; Hypoxia-Ischemia, Brain/therapy* ; Myelin Basic Protein/analysis*

The oxytocin receptor (OXTR) has garnered increasing attention for its role in regulating both mature behaviors and brain development. It has been established that OXTR mediates a range of effects that are region-specific or period-specific. However, the current studies of OXTR expression patterns in mice only provide limited help due to limitations in resolution. Therefore, our objective was to generate a comprehensive, high-resolution spatiotemporal expression map of Oxtr mRNA across the entire developing mouse brain. We applied RNAscope in situ hybridization to investigate the spatiotemporal expression pattern of Oxtr in the brains of male mice at six distinct postnatal developmental stages (P7, P14, P21, P28, P42, P56). We provide detailed descriptions of Oxtr expression patterns in key brain regions, including the cortex, basal forebrain, hippocampus, and amygdaloid complex, with a focus on the precise localization of Oxtr⁺ cells and the variance of expression between different neurons. Furthermore, we identified some neuronal populations with high Oxtr expression levels that have been little studied, including glutamatergic neurons in the ventral dentate gyrus, Vgat⁺Oxtr⁺ cells in the basal forebrain, and GABAergic neurons in layers 4/5 of the cortex. Our study provides a novel perspective for understanding the distribution of Oxtr and encourages further investigations into its functions.
Animals ; Receptors, Oxytocin/metabolism* ; Male ; Brain/growth & development* ; Mice ; Mice, Inbred C57BL ; Neurons/metabolism* ; Single-Cell Analysis ; Gene Expression Regulation, Developmental ; RNA, Messenger/metabolism* ; Animals, Newborn

OBJECTIVES: To observe the reparative effects of human umbilical cord mesenchymal stem cell (hUC-MSC) transplantation on white matter injury (WMI) in neonatal rats and explore its mechanism through the nuclear factor-kappa B (NF-κB) signaling pathway mediated by microglial cells. METHODS: Sprague-Dawley rats, aged 2 days, were randomly divided into three groups: sham-operation,WMI, and hUC-MSC (n=18 each). Fourteen days after modeling, hematoxylin-eosin staining was used to observe pathological changes in the white matter, and immunofluorescence staining was used to measure the expression level of ionized calcium-binding adapter molecule 1 (Iba1). Western blotting was used to measure the protein expression levels of inhibitory subunit of nuclear factor-kappa B alpha (IκBα), phosphorylated IκBα (p-IκBα), phosphorylated NF-κB p65 (p-NF-κB p65), myelin basic protein (MBP), and neuron-specific nuclear protein (NeuN). Quantitative real-time PCR was used to assess the mRNA expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), MBP, and NeuN. Immunohistochemistry was used to measure the protein expression levels of MBP and NeuN. On day 28, the Morris water maze test was used to evaluate spatial cognitive ability. RESULTS: Fourteen days after modeling, the sham-operation group exhibited intact white matter structure with normal cell morphology and orderly nerve fiber arrangement. In the WMI group, large-scale cell degeneration and necrosis were observed, and nerve fiber arrangement was disordered. The hUC-MSC group showed relatively normal cell morphology and more orderly nerve fibers. Compared with the sham-operation group, the WMI group had significantly higher proportions of Iba1-positive cells, increased protein levels of p-IκBα and p-NF-κB p65, and higher mRNA levels of TNF-α and IL-1β. The protein expression of IκBα and the positive expression of MBP and NeuN, as well as their protein and mRNA levels, were significantly reduced in the WMI group (P<0.05). Compared with the WMI group, the hUC-MSC group showed reduced proportions of Iba1-positive cells, decreased protein levels of p-IκBα and p-NF-κB p65, and lower mRNA levels of TNF-α and IL-1β. Furthermore, IκBα protein expression and MBP and NeuN expression (both at the protein and mRNA levels) were significantly increased in the hUC-MSC group (P<0.05). On day 28, the Morris water maze results showed that compared with the sham-operation group, the WMI group had significantly longer escape latency and fewer platform crossings (P<0.05). In contrast, the hUC-MSC group had significantly shorter escape latency and more platform crossings than the WMI group (P<0.05). CONCLUSIONS hUC-MSC transplantation can repair WMI in neonatal rats, promote the maturation of oligodendrocytes, and support neuronal survival, likely by inhibiting activation of the NF-κB signaling pathway mediated by microglial cells.
Animals ; Rats, Sprague-Dawley ; White Matter/metabolism* ; Rats ; Signal Transduction ; Mesenchymal Stem Cell Transplantation ; Humans ; NF-kappa B/metabolism* ; Animals, Newborn ; Umbilical Cord/cytology* ; Male ; NF-KappaB Inhibitor alpha/metabolism* ; I-kappa B Proteins/genetics* ; Microfilament Proteins/analysis* ; Calcium-Binding Proteins/metabolism* ; Female

OBJECTIVE: To investigate the effects of salvianolic acid A (SAA) on cardiomyocyte apoptosis and mitochondrial dysfunction in response to hypoxia/reoxygenation (H/R) injury and to determine whether the Akt signaling pathway might play a role. METHODS: An in vitro model of H/R injury was used to study outcomes on primary cultured neonatal rat cardiomyocytes. The cardiomyocytes were treated with 12.5, 25, 50 μg/mL SAA at the beginning of hypoxia and reoxygenation, respectively. Adenosine triphospate (ATP) and reactive oxygen species (ROS) levels were assayed. Cell apoptosis was evaluated by flow cytometry and the expression of cleaved-caspase 3, Bax and Bcl-2 were detected by Western blotting. The effects of SAA on mitochondrial dysfunction were examined by determining the mitochondrial membrane potential (△Ψm) and mitochondrial permeability transition pore (mPTP), followed by the phosphorylation of Akt (p-Akt) and GSK-3β (p-GSK-3β), which were measured by Western blotting. RESULTS: SAA significantly preserved ATP levels and reduced ROS production. Importantly, SAA markedly reduced the number of apoptotic cells and decreased cleaved-caspase 3 expression levels, while also reducing the ratio of Bax/Bcl-2. Furthermore, SAA prevented the loss of △Ψm and inhibited the activation of mPTP. Western blotting experiments further revealed that SAA significantly increased the expression of p-Akt and p-GSK-3β, and the increase in p-GSK-3β expression was attenuated after inhibition of the Akt signaling pathway with LY294002. CONCLUSION SAA has a protective effect on cardiomyocyte H/R injury; the underlying mechanism may be related to the preservation of mitochondrial function and the activation of the Akt/GSK-3β signaling pathway.
Adenosine Triphosphate ; analysis ; Animals ; Animals, Newborn ; Caffeic Acids ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Glycogen Synthase Kinase 3 beta ; physiology ; Lactates ; pharmacology ; Mitochondria, Heart ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; drug effects ; Myocytes, Cardiac ; drug effects ; Proto-Oncogene Proteins c-akt ; physiology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; physiology

OBJECTIVETo study the expression of SUMO-modified CCAAT enhancer binding protein α (C/EBPα) in preterm rat model of bronchopulmonary dysplasisa (BPD) induced by hyperoxia exposure and its role.

METHODSEighteen preterm rats were randomly divided into an air group and a hyperoxia group (n=9 each). The model of BPD was prepared in preterm rats exposed to hyperoxia. The rats from the two groups were sacrificed on postnatal days 4, 7 and 14 respectively (3 rats at each time) and lung tissues were harvested. Periodic acid-Schiff (PAS) staining was used to observe the differentiation of rat lung tissues. Ki67 expression was detected by immunohistochemistry. Western blot was used to measure the protein expression of small ubiquitin-related modifier-1(SUMO1) and C/EBPα. A co-immunoprecipitation assay was performed to measure the protein expression of SUMO-modified C/EBPα.

RESULTSCompared with the air group, the hyperoxia group showed a decreased glycogen content in the lung tissue on postnatal day 4, and an increased content on postnatal days 7 and 14. Over the time of hyperoxia exposure, the hyperoxia group showed an increased expression of Ki67 in the lung tissue compared with the air group at all time points. Compared with the air group, the protein expression of C/EBPα increased on postnatal day 4 and decreased on postnatal days 7 and 14 in the hyperoxia group (P<0.05). The hyperoxia group had significantly upregulated expression of SUMO1 and SUMO-modified C/EBPα compared with the air group at all time points (P<0.05). In the hyperoxia group, the protein expression of SUMO-modified C/EBPα was positively correlated with the glycogen content (r=0.529, P<0.05) and the expression of Ki67 (r=0.671, P<0.05).

CONCLUSIONSHyperoxia may induce over-proliferation and differentiation disorders of alveolar epithelial cells in preterm rat model of BPD, possibly through an increased expression of SUMO-modified C/EBP&alpha.

Animals ; Animals, Newborn ; Bronchopulmonary Dysplasia ; etiology ; metabolism ; pathology ; CCAAT-Enhancer-Binding Protein-alpha ; metabolism ; Cell Proliferation ; Disease Models, Animal ; Hyperoxia ; complications ; pathology ; Ki-67 Antigen ; analysis ; Pulmonary Alveoli ; pathology ; Rats ; Rats, Sprague-Dawley ; Sumoylation

OBJECTIVETo investigate the effect of ceftriaxone on the intestinal epithelium and microbiota in mice in the early-life stage, as well as the recovery of the intestinal epithelium and reconstruction of intestinal microbiota in adult mice.

METHODSA total of 36 BALB/C neonatal mice were randomly divided into control group and experimental group, with 18 mice in each group. The mice in the experimental group were given ceftriaxone 100 mg/kg every day by gavage within 21 days after birth. Those in the control group were given an equal volume of normal saline by gavage. Immunohistochemistry was used to measure the expression of Ki67, Muc2, and ZO-1 in the intestinal epithelium. qPCR and next-generation sequencing were used to analyze the overall concentration and composition of fecal bacteria.

RESULTSAfter 21 days of ceftriaxone intervention, the experimental group had a significant reduction in body weight, a significant reduction in the expression of Ki67 and ZO-1 and a significant increase in the expression of Muc2 in intestinal epithelial cells, a significant reduction in the overall concentration of fecal bacteria, and a significant increase in the diversity of fecal bacteria compared with the control group (P<0.05). Firmicutes was the most common type of fecal bacteria in the experimental group, and there were large amounts of Staphylococcus and Enterococcus. The experimental group had a certain degree of recovery of the intestinal epithelium, but there were still significant differences in body weight and the structure of intestinal microbiota between the two groups at 56 days after birth (P<0.05).

CONCLUSIONSEarly ceftriaxone intervention significantly affects the development of the intestinal epithelium and the construction of intestinal microbiota in the early-life stage. The injury of the intestinal microbiota in the early-life stage may continue to the adult stage and affect growth and development and physiological metabolism.

Animals ; Animals, Newborn ; Anti-Bacterial Agents ; pharmacology ; Ceftriaxone ; pharmacology ; Female ; Gastrointestinal Microbiome ; drug effects ; Intestinal Mucosa ; drug effects ; Ki-67 Antigen ; analysis ; Mice ; Mice, Inbred BALB C ; Mucin-2 ; analysis ; Zonula Occludens-1 Protein ; analysis

OBJECTIVETo investigate the protective effect of prostaglandin E1 (PGE-1) against brain injury induced by hyperoxia in neonatal rats and observe the changes in the expression of glucose-regulated protein 78 (GRP78) and cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), and to provide a theoretical basis for the clinical application of PGE-1 in the treatment of neonatal brain injury induced by hyperoxia.

METHODSSixty neonatal Wistar rats were randomly divided into air control group, hyperoxic brain injury model group, and hyperoxic brain injury+PGE-1 group. All rats except those in the air control group were treated to establish a hyperoxic brain injury model. From the first day of modeling, the rats in the hyperoxia brain injury+PGE-1 group were intraperitoneally injected with PGE-1 2 μg/kg daily for 7 consecutive days, while the other two groups were treated with normal saline instead. The water content of brain tissue was measured; the pathological changes of brain tissue were evaluated by hematoxylin-eosin staining; the apoptosis of brain cells was assessed by nuclear staining combined with TUNEL staining; the protein expression of GRP78 and CHOP in brain tissue was measured by Western blot.

RESULTSThe water content of brain tissue in the hyperoxic brain injury model group was significantly higher than that in the hyperoxic brain injury+PGE-1 group and air control group (P<0.05); the water content of brain tissue in the hyperoxic brain injury+PGE-1 group was significantly higher than that in the air control group (P<0.05). The pathological section of brain tissue showed inflammatory cell infiltration and mild cerebrovascular edema in the brain parenchyma in the hyperoxic brain injury model group; the periparenchymal inflammation and edema in the hyperoxic brain injury+PGE-1 group were milder than those in the hyperoxic brain injury model group. The apoptosis index of brain tissue in the hyperoxic brain injury model group was significantly higher than that in the hyperoxic brain injury+PGE-1 group and air control group (P<0.05); the apoptosis index of brain tissue in the hyperoxic brain injury+PGE-1 group was significantly higher than that in the air control group (P<0.05). The protein expression of GRP78 and CHOP in brain tissue was significantly higher in the hyperoxic brain injury model group than in the hyperoxic brain injury+PGE-1 group and air control group (P<0.05); the protein expression of GRP78 and CHOP was significantly higher in the hyperoxic brain injury+PGE-1 group than in the air control group (P<0.05).

CONCLUSIONSPGE-1 has a protective effect against hyperoxia-induced brain injury in neonatal rats, which may be related to the inhibition of cell apoptosis by down-regulating the expression of GRP78 and CHOP.

Alprostadil ; therapeutic use ; Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Brain ; pathology ; Brain Injuries ; metabolism ; pathology ; prevention & control ; Heat-Shock Proteins ; analysis ; Hyperoxia ; complications ; Neuroprotective Agents ; therapeutic use ; Rats ; Rats, Wistar ; Transcription Factor CHOP ; analysis

OBJECTIVETo study the effect of Bifidobacterium on the expression of β-defensin-2 (BD-2) in intestinal tissue of neonatal rats with necrotizing enterocolitis (NEC).

METHODSA total of 40 rats were randomly divided into four groups: normal control, Bifidobacterium control, NEC model, and Bifidobacterium treatment, with 10 rats in each group. A rat model of NEC was induced by hypoxia, cold stimulation, and artificial feeding. The rats in the Bifidobacterium control and Bifidobacterium treatment groups were given Bifidobacterium via the gastric tube after cold stimulation once a day for three consecutive days. The morphological changes of the terminal ileum were observed under a light microscope and the intestinal injury score was determined. Immunohistochemistry and qRT-PCR were used to measure the protein and mRNA expression of BD-2 in the ileal mucosal tissue.

RESULTSThe NEC model group had a significantly higher intestinal injury score than the normal control, Bifidobacterium control, and Bifidobacterium treatment groups (P<0.05). The Bifidobacterium treatment group had a significantly higher intestinal injury score than the normal control and Bifidobacterium control groups (P<0.05). The mRNA and protein expression of BD-2 in the normal control group was significantly lower than in the Bifidobacterium control, NEC model, and Bifidobacterium treatment groups (P<0.05). The Bifidobacterium control group had significantly higher mRNA and protein expression of BD-2 than the NEC model and Bifidobacterium treatment groups (P<0.05). The Bifidobacterium treatment group had significantly higher mRNA and protein expression of BD-2 than the NEC model group (P<0.05).

CONCLUSIONSBifidobacterium can induce the expression of BD-2 in intestinal tissue of rats and reduce inflammatory response by increasing the expression of BD-2. This provides a protective effect on neonatal rats with NEC.

Animals ; Bifidobacterium ; Disease Models, Animal ; Enterocolitis, Necrotizing ; therapy ; Humans ; Infant, Newborn ; Intestinal Mucosa ; metabolism ; NF-kappa B ; physiology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; physiology ; beta-Defensins ; analysis ; genetics ; physiology

OBJECTIVETo explore the effects of rat bone mesenchymal stem cell (BMSC) transplantation on retinal neovascularization, and to observe the changes of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factors (VEGF) in rats with oxygen-induced retinopathy (OIR).

METHODSSeventy-two seven-day-old Sprague-Dawley rats were randomly divided into three groups: normal control (CON), model (OIR) and BMSC transplantation. In the BMSC transplantation group, BMSCs were transplanted 5 days after oxygen conditioning. The phosphate buffered saline of the same volume was injected in the CON and OIR groups. The OIR model was prerpared according to the classic hyperoxygen method. At seven days after transplantation, retinal neovascularization was examined by retinal flat-mount staining and hematoxylin eosin (HE) staining. The expression of HIF-1α and VEGF proteins was examined by immunohistochemistry staining and Western blot analysis.

RESULTSThe retinal flat-mount staining results showed that the vessels were well organized in the CON group, but the vessels were irregularly organized, and lots of nonperfusion areas were observed in the OIR group. The large vessels were a bit circuitous, the retinal vessels were relatively organized, and less nonperfusion areas were noted in the BMSC transplantation group. The HE staining results showed that many neovessels and preretinal neovascular (pre-RNC) cells were observed on the internal limiting membrane in the OIR group. There were less pre-RNC cells in the BMSC transplantation group compared with the OIR group (P<0.01). The immunohistochemistry analysis showed that more HIF-1αand VEGFcells were observed in the OIR group compared with the CON group, and less HIF-1αand VEGFcells were observed in the BMSC transplantation group compared with OIR group (P<0.05). The Western blot analysis showed the expression of HIF-1α and VEGF proteins in the OIR group was significantly higher than that in the CON group. The expression of HIF-1α and VEGF proteins in the BMSC transplantation group was lower than that in the OIR group (P<0.01).

CONCLUSIONSBMSC transplantation therapy could alleviate retinal neovascularization in OIR rats, and its mechanisms might be associated with the inhibition of the expression of HIF-1α and VEGF proteins.

Animals ; Animals, Newborn ; Female ; Hypoxia-Inducible Factor 1, alpha Subunit ; analysis ; Male ; Mesenchymal Stem Cell Transplantation ; Rats ; Rats, Sprague-Dawley ; Retina ; chemistry ; Retinal Neovascularization ; prevention & control ; Retinopathy of Prematurity ; metabolism ; therapy ; Vascular Endothelial Growth Factor A ; analysis