OBJECTIVE: To illustrate the role of dehydrocorydaline (DHC) in chronic constriction injury (CCI)-induced neuropathic pain and the underlying mechanism. METHODS: C57BL/6J mice were randomly divided into 3 groups by using a random number table, including sham group (sham operation), CCI group [intrathecal injection of 10% dimethyl sulfoxide (DMSO)], and CCI+DHC group (intrathecal injection of DHC), 8 mice in each group. A CCI mouse model was conducted to induce neuropathic pain through ligating the right common sciatic nerve. On day 14 after CCI modeling or sham operation, mice were intrathecal injected with 5 µL of 10% DMSO or 10 mg/kg DHC (5 µL) into the 5th to 6th lumbar intervertebral space (L5-L6). Pregnant ICR mice were sacrificed for isolating primary spinal neurons on day 14 of embryo development for in vitro experiment. Pain behaviors were evaluated by measuring the paw withdrawal mechanical threshold (PWMT) of mice. Immunofluorescence was used to observe the activation of astrocytes and microglia in mouse spinal cord. Protein expressions of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), phosphorylation of N-methyl-D-aspartate receptor subunit 2B (p-NR2B), and NR2B in the spinal cord or primary spinal neurons were detected by Western blot. RESULTS: In CCI-induced neuropathic pain model, mice presented significantly decreased PWMT, activation of glial cells, overexpressions of iNOS, TNF-α, IL-6, and higher p-NR2B/NR2B ratio in the spinal cord (P<0.05 or P<0.01), which were all reversed by a single intrathecal injection of DHC (P<0.05 or P<0.01). The p-NR2B/NR2B ratio in primary spinal neurons were also inhibited after DHC treatment (P<0.05). CONCLUSION An intrathecal injection of DHC relieved CCI-induced neuropathic pain in mice by inhibiting the neuroinflammation and neuron hyperactivity.
Animals ; Neuralgia/etiology* ; Mice, Inbred C57BL ; Analgesics/pharmacology* ; Neuroinflammatory Diseases/pathology* ; Constriction ; Male ; Receptors, N-Methyl-D-Aspartate/metabolism* ; Nitric Oxide Synthase Type II/metabolism* ; Mice, Inbred ICR ; Microglia/pathology* ; Spinal Cord/drug effects* ; Female ; Mice ; Tumor Necrosis Factor-alpha/metabolism* ; Disease Models, Animal ; Constriction, Pathologic/complications* ; Interleukin-6/metabolism* ; Astrocytes/metabolism* ; Chronic Disease ; Neurons/metabolism*

OBJECTIVE: To investigate whether Buyang Huanwu Decoction (BYHWD) had a good curative effect on the neuroprotection of red nucleus neurons after spinal cord injury (SCI) and the possible molecular mechanism. METHODS: Ninety male Sprague-Dawley rats were divided into 5 groups (n=18 per group) according to a random number table, including the control, model, low- (12.78 g/kg, BL group), medium- (25.65 g/kg, BM group), and high-dose BYHWD groups (51.30 g/kg, BH group). A rubrospinal tract transection model in rats was established, and different doses of BYHWD were intragastrically administrated for 4 weeks. The forelimb locomotor function was recorded using the spontaneous vertical exploration test. Cyclic adenosine monophosphate (cAMP) level in red nucleus was detected through an enzyme-linked immunosorbent assay. The morphology and number of red nucleus neurons were observed using Nissl's staining and axonal retrograde tracing by Fluoro-Gold (FG). The expression of cAMP-dependent protein kinase A (PKA), nuclear factor kappa-B (NF-κB) p65, and brain-derived neurotrophic factor (BDNF) in red nucleus were detected using immunohistochemistry and quantitative real-time polymerase chain reaction. RESULTS: Compared with the control group, the utilization rate of bilateral forelimbs, unilateral right forelimbs, proportion of FG-labeled positive neurons, cAMP level, protein expressions of PKA and BDNF, and BDNF mRNA expression were significantly decreased in the model group (P<0.01), while NF-κB p65 was increased in the model group (P<0.01). Compared with the model group, the utilization rate of bilateral forelimbs and unilateral right forelimbs were significantly higher in the BL, BM and BH groups (P<0.01), the proportion of FG-labeled positive neurons, cAMP level, protein expressions of PKA and BDNF and BDNF mRNA expression in all BYHWD groups were increased (P<0.05 or P<0.01), while NF-κB p65 were decreased in all BYHWD groups (P<0.05 or P<0.01). CONCLUSIONS BYHWD possesses a sound neuroprotective effect on red nucleus neurons after SCI, and the efficacy was dose-related. The mechanism may be related to regulating the cAMP/PKA/NF-κ B p65 signaling pathway, finally promoting expression of BDNF.
Animals ; Spinal Cord Injuries/pathology* ; Drugs, Chinese Herbal/therapeutic use* ; Rats, Sprague-Dawley ; Male ; Cyclic AMP/metabolism* ; Transcription Factor RelA/metabolism* ; Cyclic AMP-Dependent Protein Kinases/metabolism* ; Signal Transduction/drug effects* ; Brain-Derived Neurotrophic Factor/genetics* ; Red Nucleus/metabolism* ; Recovery of Function/drug effects* ; Neurons/metabolism* ; Rats

OBJECTIVES: The neurotoxicity of carbon monoxide (CO) to the central nervous system is a key pathogenesis of delayed encephalopathy after acute carbon monoxide poisoning (DEACMP). Our previous study found that retinoic acid (RA) can suppress the neurotoxic effects of CO. This study further explores, in vivo and in vitro, the molecular mechanisms by which RA alleviates CO-induced central nervous system damage. METHODS: A cytotoxic model was established using the mouse hippocampal neuronal cell line HT22 and primary oligodendrocytes exposed to CO, and a DEACMP animal model was established in adult Kunming mice. Cell viability and apoptosis of hippocampal neurons and oligodendrocytes were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Annexin V/propidium iodide (PI) double staining. The transcriptional and protein expression of each gene was detected using real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting. Long noncoding RNA (lncRNA) SNHG15 and LINGO-1 were knocked down or overexpressed to observe changes in neurons and oligodendrocytes. In DEACMP mice, SNHG15 or LINGO-1 were knocked down to assess changes in central nervous tissue and downstream protein expression. RESULTS: RA at 10 and 20 μmol/L significantly reversed CO-induced apoptosis of hippocampal neurons and oligodendrocytes, downregulation of SNHG15 and LINGO-1, and upregulation of brain-derived neurotrophic factor (BDNF) and tyrosine kinase receptor B (TrkB) (all P<0.05). Overexpression of SNHG15 or LINGO-1 weakened the protective effect of RA against CO-induced cytotoxicity (all P<0.05). Knockdown of SNHG15 or LINGO-1 alleviated CO-induced apoptosis of hippocampal neurons and oligodendrocytes and upregulated BDNF and TrkB expression levels (all P<0.05). Experiments in DEACMP model mice showed that knockdown of SNHG15 or LINGO-1 mitigated central nervous system injury in DEACMP (all P<0.05). CONCLUSIONS RA alleviates CO-induced apoptosis of hippocampal neurons and oligodendrocytes, thereby reducing central nervous system injury and exerting neuroprotective effects. LncRNA SNHG15 and LINGO-1 are key molecules mediating RA-induced inhibition of neuronal apoptosis and are associated with the BDNF/TrkB pathway. These findings provide a theoretical framework for optimizing the clinical treatment of DEACMP and lay an experimental foundation for elucidating its molecular mechanisms.
Animals ; RNA, Long Noncoding/physiology* ; Brain-Derived Neurotrophic Factor/genetics* ; Carbon Monoxide Poisoning/complications* ; Mice ; Tretinoin/pharmacology* ; Nerve Tissue Proteins/metabolism* ; Membrane Proteins/metabolism* ; Apoptosis/drug effects* ; Hippocampus/cytology* ; Receptor, trkB/metabolism* ; Neurons/drug effects* ; Male ; Brain Diseases/etiology* ; Oligodendroglia/drug effects* ; Signal Transduction ; Cell Line

OBJECTIVES: To investigate the effect of monotropein on motor function recovery of mice with spinal cord injury (SCI) and explore the underlying mechanism. METHODS: Forty-five adult female C57BL/6 mice were randomized equally into sham operation group, SCI group, and SCI group with daily intraperitoneal monotropein injection. The mice in the former two groups received daily saline injections. Motor function of the mice was evaluated using BMS scores, slant plate test, and footprint analyses. Pathological changes and neuronal counts in the spinal cord were observed using HE, LFB, and Nissl staining. The biological functions of monotropein were explored using GO and KEGG enrichment analyses. NeuN/cleaved caspase-3 immunofluorescence assay and Western blotting were used to detect neuronal apoptosis in the spinal cord of the mice. In cultured HT22 cells, the effect of monotropein on TNF-α-induced cell apoptosis was evaluated using TUNEL staining and Western blotting. In monotropein-treated HT22 cells and SCI mice, the changes in the PI3K/AKT pathway were examined, and the effect of a PI3K/AKT pathway activator (IGF-1) on HT22 cell apoptosis and motor function recovery of SCI mice were observed. RESULTS: SCI mice with monotropein treatment showed significantly improved motor functions with reduced SCI areas and increased myelin retention and neuron counts in the spinal cord. Bioinformatics analysis suggested a role of PI3K/AKT signaling pathway in mediating the anti-apoptotic effects of monotropein. In SCI mice, monotropein obviously reduced apoptotic neurons, decreased expressions of cleaved caspase-3 and Bax and increased Bcl-2 expression in the spinal cord. In HT22 cells, monotropein significantly inhibited TNF-α-induced apoptosis and PI3K/AKT pathway activation. Treatment with IGF-1 obviously increased apoptosis of HT22 cells and exacerbated locomotor dysfunction in SCI mice. CONCLUSIONS Monotropein promotes motor function recovery in SCI mice by reducing neuronal apoptosis possibly by inhibiting the PI3K/AKT signaling pathway.
Animals ; Spinal Cord Injuries/metabolism* ; Apoptosis/drug effects* ; Signal Transduction/drug effects* ; Mice, Inbred C57BL ; Mice ; Proto-Oncogene Proteins c-akt/metabolism* ; Female ; Phosphatidylinositol 3-Kinases/metabolism* ; Neurons/pathology* ; Recovery of Function

OBJECTIVES: To explore whether the antioxidant axis Nrf2/HO-1 is involved in the regulation of hippocampus injury induced by cypermethrin and its underlying mechanism. METHODS: Ten-week-old C57BL/6 mice were randomly divided into control group and cypermethrin exposure groups with low, medium, and high exposure levels. After 21 days of oral gavage of corn oil (control) or cypermethrin, the levels of MDA, T-SOD, GSH-Px and CAT in the hippocampus of the mice were examined to evaluate the oxidative stress levels. HE staining was used to observe morphological changes of the hippocampal neurons. Western blotting, immunofluorescence staining and RT-qPCR were employed to detect the protein expressions and mRNA expression of Nrf2 and HO-1 and HO-1. RESULTS: Subacute oral exposure to cypermethrin significantly increased MDA level, decreased the activities of antioxidant enzymes T-SOD, GSH-Px and CAT, and induced neuronal damage in the CA1 and CA3 regions in the hippocampus of C57BL/6 mice. Cypermethrin exposure also caused Nrf2 protein translocation from the cytoplasm to the nucleus, accompanied by upregulated expression levels of the key antioxidant factor Nrf2 and its downstream target kinase HO-1. CONCLUSIONS Cypermethrin exposure dose-dependently causes oxidative damage in the hippocampus of C57BL/6 mice, which is regulated by the Nrf2/HO-1 antioxidant pathway.
Animals ; Pyrethrins/toxicity* ; NF-E2-Related Factor 2/metabolism* ; Hippocampus/cytology* ; Mice, Inbred C57BL ; Mice ; Oxidative Stress/drug effects* ; Neurons/pathology* ; Heme Oxygenase-1/metabolism* ; Signal Transduction ; Membrane Proteins

OBJECTIVES: To explore the effects of cannabidiol on endoplasmic reticulum stress and neuronal apoptosis in rats with multiple concussions (MCC). METHODS: SD rats were randomized into sham group, MCC group, 1% tween20 (TW) treatment group, and low-dose (10 mg/kg) and high-dose (40 mg/kg) cannabidiol treatment groups. In all but the sham group, MCC models were established using a metal pendulum percussion device, after which the rats received daily intraperitoneal injections of the corresponding agents for 2 weeks. The expressions of PERK, eIF2α, ATF4, CHOP, TRIB3, p-Akt and pro-caspase-3 in the brain tissue of the rats were detected with qRT-PCR, Western blotting and immunofluorescence staining. The core targets of cannabidiol in treatment of traumatic brain injury (TBI) were identified by network pharmacology analysis, and molecular docking was carried out to simulate the interaction of cannabidiol with the factors related to endoplasmic reticulum stress and apoptosis. RESULTS: Compared with the sham-operated rats, the rat models of MCC showed significantly increased mRNA expressions of PERK, eIF2α and CHOP and protein expressions of PERK, eIF2α, ATF4, CHOP, TRIB3, p-AKT and pro-caspase-3 in the cerebral cortex. CBD treatment, especially at the high dose, obviously increased the expression of p-Akt and lowered the expression levels of the other factors tested in the rat models. Network pharmacology analysis indicated interactions of the core targets of CBD with the factors related to endoplasmic reticulum stress and TBI, and molecular docking study showed a high binding energy of CBD with multiple factors pertaining to endoplasmic reticulum stress and apoptosis. CONCLUSIONS MCC induce endoplasmic reticulum stress and apoptosis in rat brain tissues, for which CBD, especially at a high dose, provides neuroprotective effects by inhibiting endoplasmic reticulum stress and cell apoptosis.
Animals ; Endoplasmic Reticulum Stress/drug effects* ; Apoptosis/drug effects* ; Rats, Sprague-Dawley ; Activating Transcription Factor 4/metabolism* ; Transcription Factor CHOP/metabolism* ; Rats ; Eukaryotic Initiation Factor-2/metabolism* ; Signal Transduction/drug effects* ; eIF-2 Kinase/metabolism* ; Cannabidiol/pharmacology* ; Neurons/metabolism* ; Brain Concussion/metabolism* ; Male ; Molecular Docking Simulation

OBJECTIVES: To investigate the subcellular localization and biological functions of transmembrane protein 240 (TMEM240). METHODS: NCBI BLAST and TMHMM bioinformatics software were used for protein sequence analysis and prediction of transmembrane domain of TMEM240. Brain tissues from male C57BL/6 mice (18-20 days old) were examined for distribution of TMEM240 using in situ hybridization, and qPCR and Western blotting were used to detect TMEM240 expression in different mouse tissues and in cortical neurons at different time points (n=3). In the in vitro experiment, HepG2 and Neuro-2a cells were transfected with plasmids for overexpression of TMEM240, and subcellular localization of TMEM240 was analyzed using cell imaging. In primary cultures of cortical neurons isolated from C57BL/6 mice, TMEM240 expression and its biological functions were investigated using qPCR, Western blotting, and immunofluorescence staining. RESULTS: Human and mouse TMEM240 proteins share a 97.69% similarity in the protein sequences, and both are transmembrane proteins with two transmembrane domains. TMEM240 mRNA and protein were highly expressed in mouse brain tissues and cortical neurons. In isolated mouse cortical neurons, TMEM240 expression reached the peak level after primary culture for 9 days and distributed in scattered spots within the cells. In HepG2 cells, TMEM240 was characterized as intracellular membrane structures and showed 80% colocalization with peroxisomes. In Neuro-2a cells, TMEM240 overexpression caused significant enhancement of the expressions of Rho GDP dissociation inhibitor β (ARHGDIB) at both the mRNA and protein levels. CONCLUSIONS TMEM240 is a novel intracellular subcellular structure specifically expressed in neurons with significant potential for targeted cellular function regulation.
Animals ; Humans ; Mice ; Peroxisomes/metabolism* ; Membrane Proteins/genetics* ; Mice, Inbred C57BL ; Neurons/metabolism* ; Male ; rho-Specific Guanine Nucleotide Dissociation Inhibitors ; Hep G2 Cells ; Brain/metabolism*

Autism Spectrum Disorders (ASDs) are reported as a group of neurodevelopmental disorders. The structural changes of brain regions including the hippocampus were widely reported in autistic patients and mouse models with dysfunction of ASD risk genes, but the underlying mechanisms are not fully understood. Here, we report that deletion of Trio, a high-susceptibility gene of ASDs, causes a postnatal dentate gyrus (DG) hypoplasia with a zigzagged suprapyramidal blade, and the Trio-deficient mice display autism-like behaviors. The impaired morphogenesis of DG is mainly caused by disturbing the postnatal distribution of postmitotic granule cells (GCs), which further results in a migration deficit of neural progenitors. Furthermore, we reveal that Trio plays different roles in various excitatory neural cells by spatial transcriptomic sequencing, especially the role of regulating the migration of postmitotic GCs. In summary, our findings provide evidence of cellular mechanisms that Trio is involved in postnatal DG morphogenesis.
Animals ; Dentate Gyrus/metabolism* ; Mice ; Morphogenesis/physiology* ; Neurons/pathology* ; Cell Movement ; Mice, Inbred C57BL ; Autism Spectrum Disorder/pathology* ; Mice, Knockout ; Neural Stem Cells ; Male ; Neurogenesis ; Autistic Disorder/genetics*

Stromal interaction molecules (STIM)s are Ca²⁺ sensors in internal Ca²⁺ stores of the endoplasmic reticulum. They activate the store-operated Ca²⁺ channels, which are the main source of Ca²⁺ entry in non-excitable cells. Moreover, STIM proteins interact with other Ca²⁺ channel subunits and active transporters, making STIMs an important intermediate molecule in orchestrating a wide variety of Ca²⁺ influxes into excitable cells. Nevertheless, little is known about the role of STIM proteins in brain functioning. Being involved in many signaling pathways, STIMs replenish internal Ca²⁺ stores in neurons and mediate synaptic transmission and neuronal excitability. Ca²⁺ dyshomeostasis is a signature of many pathological conditions of the brain, including neurodegenerative diseases, injuries, stroke, and epilepsy. STIMs play a role in these disturbances not only by supporting abnormal store-operated Ca²⁺ entry but also by regulating Ca²⁺ influx through other channels. Here, we review the present knowledge of STIMs in neurons and their involvement in brain pathology.
Neurons/metabolism* ; Animals ; Humans ; Calcium/metabolism* ; Stromal Interaction Molecules/metabolism* ; Calcium Signaling/physiology* ; Calcium Channels/metabolism* ; Brain/metabolism*

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