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*

BACKGROUNDThe interaction between activated microglia and T lymphocytes can yield abundant pro-inflammatory cytokines. Our previous study proved that thymus immune tolerance could alleviate the inflammatory response. This study aimed to investigate whether intrathymic injection of myelin basic protein (MBP) in mice could suppress the inflammatory response after co-culture of T lymphocytes and BV-2 microglia cells.

METHODSTotally, 72 male C57BL/6 mice were randomly assigned to three groups (n = 24 in each): Group A: intrathymic injection of 100 μl MBP (1 mg/ml); Group B: intrathymic injection of 100 μl phosphate-buffered saline (PBS); and Group C: sham operation group. Every eight mice in each group were sacrificed to obtain the spleen at postoperative days 3, 7, and 14, respectively. T lymphocytes those were extracted and purified from the spleens were then co-cultured with activated BV-2 microglia cells at a proportion of 1:2 in the medium containing MBP for 3 days. After identified the T lymphocytes by CD3, surface antigens of T lymphocytes (CD4, CD8, CD152, and CD154) and BV-2 microglia cells (CD45 and CD54) were detected by flow cytometry. The expressions of pro-inflammatory factors of BV-2 microglia cells (interleukin [IL]-1β, tumor necrosis factor-α [TNF-α], and inducible nitric oxide synthase [iNOS]) were detected by quantitative real-time polymerase chain reaction (PCR). One-way analysis of variance (ANOVA) and the least significant difference test were used for data analysis.

RESULTSThe levels of CD152 in Group A showed an upward trend from the 3rd to 7th day, with a downward trend from the 7th to 14th day (20.12 ± 0.71%, 30.71 ± 1.14%, 13.50 ± 0.71% at postoperative days 3, 7, and 14, respectively, P < 0.05). The levels of CD154 in Group A showed a downward trend from the 3rd to 7th day, with an upward trend from the 7th to 14th day (10.00 ± 0.23%, 5.28 ± 0.69%, 14.67 ± 2.71% at postoperative days 3, 7, and 14, respectively, P < 0.05). The ratio of CD4+/CD8 + T in Group A showed a downward trend from the 3rd to 7th day, with the minimum at postoperative day 7, then an upward trend from the 7th to 14th day (P < 0.05). Meanwhile, the levels of CD45 and CD54 in Group A were found as the same trend as the ratio of CD4+/CD8 + T (CD45: 83.39 ± 2.56%, 82.74 ± 2.09%, 87.56 ± 2.11%; CD54: 3.80 ± 0.24%, 0.94 ± 0.40%, 3.41 ± 0.33% at postoperative days 3, 7, and 14, respectively, P < 0.05). The expressions of TNF-α, IL-1β, and iNOS in Group A were significantly lower than those in Groups B and C, and the values at postoperative day 7 were the lowest compared with those at postoperative days 3 and 14 (P < 0.05). No significant difference was found between Groups B and C.

CONCLUSIONSIntrathymic injection of MBP could suppress the immune reaction that might reduce the secondary immune injury of brain tissue induced by an inflammatory response.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Antigens, Surface ; analysis ; Brain Injuries, Traumatic ; drug therapy ; CD4-CD8 Ratio ; Coculture Techniques ; Male ; Mice ; Mice, Inbred C57BL ; Microglia ; immunology ; Myelin Basic Protein ; administration & dosage ; pharmacology ; T-Lymphocytes ; immunology

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism

OBJECTIVETo study the effects of caffeine citrate on myelin basic protein (MBP) expression in the cerebral white matter of neonatal rats with hypoxic-ischemic brain damage (HIBD) and the related mechanism.

METHODSForty-eight seven-day-old Sprague-Dawley neonatal rats were randomly assigned to 3 groups: sham operation (n=16), HIBD (n=16) and HIBD+caffeine citrate (n=16). The rats in the HIBD and HIBD+caffeine citrate groups were subjected to left common carotid artery ligation, and then were exposed to 80 mL/L oxygen and 920 mL/L nitrogen for 2 hours to induce HIBD. The rats in the sham operation group were only subjected to a sham operation, without the left common carotid artery ligation or hypoxia exposure. Caffeine citrate (20 mg/kg) was injected intraperitoneally before hypoxia ischemia (HI) and immediately, 24 hours, 48 hours and 72 hours after HI. The other two groups were injected intraperitoneally with an equal volume of normal saline at the corresponding time points. On postnatal day 12, the expression of MBP in the left subcortical white matter was detected by immunohistochemistry, and the levels of adenosine A1 receptor mRNA and A2a receptor mRNA in the left brain were detected by real-time PCR.

RESULTSThe expression of MBP in the left subcortical white matter in the HIBD group was lower than in the sham operation group (P<0.05). The MBP expression in the HIBD+caffeine citrate group was significantly higher than in the HIBD group, but was still lower than the sham operation group (P<0.05). Real-time PCR showed that the adenosine A1 receptor mRNA expression was significantly higher in the HIBD group than in the sham operation group, and it was significantly lower in the HIBD+caffeine citrate group than in the HIBD group (P<0.05).

CONCLUSIONSCaffeine citrate can improve brain white matter damage following HIBD in neonatal rats and the protection mechanism might be related with the down-regulation of adenosine A1 receptor expression.

Animals ; Animals, Newborn ; Caffeine ; pharmacology ; Citrates ; pharmacology ; Female ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; pathology ; Male ; Myelin Basic Protein ; analysis ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptor, Adenosine A1 ; genetics ; Receptor, Adenosine A2A ; genetics ; White Matter ; chemistry

OBJECTIVETo study the effects of perinatal recurrent infection on the brain development in immature mice.

METHODSSix pregnant C57BL6 mice were randomly assigned to three groups: intrauterine infection, perinatal recurrent infection and control. The intrauterine infection group was intraperitoneally injected with LPS (0.5 mg/kg) on the 18th day of pregnancy. The perinatal recurrent infection group was injected with LPS (0.5 mg/kg) on the 18th day of pregnancy and their offsprings were intraperitoneally injected with the same dose of LPS daily from postnatal day 3 to 12. The control group was administered with normal saline at the same time points as the recurrent infection group. The short-time neurobehaviors were assessed on postnatal day 13. The mice were then sacrificed to measure brain weights and neuropathological changes using cresyl violet staining. Western blot was used to evaluate the expression of TNF-α, Caspase-3 and myelin basic protein (MBP).

RESULTSThe brain weights of the recurrent infection group were significantly lower than the control and intrauterine infection groups (P<0.05) and the recurrent infection group displayed significant neuropathological changes. Perinatal recurrent infection resulted in increased expression levels of TNF-α and Caspase-3, and decreased expression level of MBP compared with the intrauterine infection and control groups (P<0.01). The neurobehavior test showed that the recurrent infection group used longer time in gait reflex, right reflex and geotaxis reflex compared with the control and intrauterine infection groups on postnatal day 13 (P<0.05).

CONCLUSIONSPerinatal recurrent infection may exacerbate inflammatory response and cell death in the immature brain, which may be one of the important factors for perinatal brain injury.

Animals ; Animals, Newborn ; Bacterial Infections ; physiopathology ; Body Weight ; Brain ; growth & development ; pathology ; Caspase 3 ; analysis ; Female ; Mice ; Mice, Inbred C57BL ; Myelin Basic Protein ; analysis ; Pregnancy ; Recurrence ; Reflex

The present study was performed to evaluate the relationship between the neurotoxicity of acrylamide and the differential gene expression pattern in mice. Both locomotor test and rota-rod test showed that the group treated with higher than 30 mg/kg/day of acrylamide caused impaired motor activity in mice. Based on cDNA microarray analysis of mouse brain, myelin basic protein gene, kinesin family member 5B gene, and fibroblast growth factor (FGF) 1 and its receptor genes were down-regulated by acrylamide. The genes are known to be essential for neurofilament synthesis, axonal transport, and neuro-protection, respectively. Interestingly, both FGF 1 and its receptor genes were down-regulated. Genes involved in nucleic acid binding such as AU RNA binding protein/enoyl-coA hydratase, translation initiation factor (TIF) 2 alpha kinase 4, activating transcription factor 2, and U2AF 1 related sequence 1 genes were down-regulated. More interesting finding was that genes of both catalytic and regulatory subunit of protein phosphatases which are important for signal transduction pathways were down-regulated. Here, we propose that acrylamide induces neurotoxicity by regulation of genes associated with neurofilament synthesis, axonal transport, neuro-protection, and signal transduction pathways.
Acrylamide ; Activating Transcription Factor 2 ; Animals ; Axonal Transport ; Brain ; Fibroblast Growth Factors ; Gene Expression ; Humans ; Kinesin ; Mice ; Motor Activity ; Myelin Basic Protein ; Oligonucleotide Array Sequence Analysis ; Peptide Initiation Factors ; Phosphoprotein Phosphatases ; Phosphotransferases ; RNA ; Signal Transduction

OBJECTIVETo study the effect of intracerebral transplantation of bone marrow stromal cells (BMSCs) on brain white matter of neonatal rats with hypoxic-ischemic brain damage (HIBD).

METHODSThirty-four 7-day-old neonatal rats were randomly assigned to three groups: normal control (n=10), HIBD (n=12) and HIBD+BMSCs transplantation (n=12). The HIBD and the HIBD+BMSCs transplantation group rats were subjected to left carotid artery ligation, followed by hypoxia exposure for 2 hrs, in order to induce HIBD. The rats in the HIBD+BMSCs transplantation group received transplantation of BMSCs labeled nucleus with Hochest 33324 into the left hippocampus 24 hrs after HIBD induction. Myelin basic protein (MBP) expression in the left corpus callosum and the subcortical white matter and the number of oligodendrocyte precursors positively stained O4 in the left periventricular area and the subcortical white matter were detected by immunohistochemistry at ages of 45 days.

RESULTSThe labeled BMSCs survived and were found mainly in the left hemisphere 37 days after transplantation. The positive rate of O4 expressed by the transplanted BMSCs was 3.70+/-1.09%. More hypomyelination in the left corpus callosum and the subcortical white matter, and less number of O4 positive oligodendrocytes in the left periventricular area and the subcortical white matter were found in the HIBD group compared with the normal control group (P<0.01). The HIBD rats receiving BMSCs transplantation had increased O4 positive oligodendrocytes in the left periventricular area and the subcortical white matter and improved MBP immunoreactivity in the left corpus callosum and the subcortical white matter compared with the HIBD group (P<0.01).

CONCLUSIONSIntracerebral transplantation of BMSCs can improve brain white matter damage in neonatal rats with HIBD.

Animals ; Animals, Newborn ; Antigens, Differentiation ; analysis ; Bone Marrow Cells ; physiology ; Brain ; pathology ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; therapy ; Immunohistochemistry ; Myelin Basic Protein ; analysis ; Rats ; Rats, Sprague-Dawley ; Stromal Cells ; transplantation

OBJECTIVEA recent study has suggested that hyperbaric oxygen (HBO) therapy administered within 3 hrs following hypoxic-ischemic brain damage (HIBD) may alleviate brain white matter damage (WMD) in neonatal rats. However it is unclear whether a delayed HBO therapy (more than 3 hrs following HIBD) has neuroprotective effects in neonatal rats. This study aimed to explore the effect of HBO therapy administered at different time points following HIBD on WMD in neonatal rats.

METHODSThe HIBD model was prepared according to the Rice-Vannucci procedure in 7-day-old Sprague-Dawley rats. HBO therapy was administered at 3, 6, 12, 24 or 72 hrs after HIBD, once daily for consecutive 7 days. T-maze test, the foot-fault test and the radial arm maze test were performed after 14 days of HIBD. Myelin basic protein (MBP) in the callositas and corpora striata was examined by immunohistochemical method 28 days after HIBD.

RESULTSThe rats receiving HBO therapy at 3, 6 and 12 hrs after HIBD performed significantly better in the T-maze test, the radial arm maze test and the foot-fault test than the untreated HIBD rats. There were no significant differences in the behavioral test results between the HBO-treated groups administered HBO at 24 and 72 hrs after HIBD and the untreated HIBD group. The MBP expression in the HBO-treated groups treated within 12 hrs after HIBD was significantly higher than that in the untreated HIBD group (P < 0.05). When the HBO therapeutic window was delayed to 24 hrs after HIBD, there were no significant differences in the MBP expression between the HBO-treated and the untreated HIBD groups.

CONCLUSIONSHBO therapy administered within 12 hrs following HIBD can alleviate brain WMD in neonatal rats, but the efficacy of HBO therapy administered 24 hrs after HIBD does not appear to be satisfactory.

Animals ; Animals, Newborn ; Brain ; pathology ; Female ; Hyperbaric Oxygenation ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; psychology ; therapy ; Immunohistochemistry ; Male ; Maze Learning ; Myelin Basic Protein ; analysis ; Rats ; Rats, Sprague-Dawley ; Time Factors

OBJECTIVEThis study investigated the effect of hyperbaric oxygenation (HBO) on neural stem cells (NSCs) and myelin in neonatal rats following hypoxic-ischemic brain damage (HIBD) and aimed to explore the possible mechanism of the protective effect of HBO on HIBD.

METHODSSeven-day-old Sprague-Dawley rat pups were randomly assigned into 4 groups: Normal control, HIBD, hyperbaric air (HBA), and HBO groups (n=30 each). The HIBD model was produced by permanent occlusion of the left common carotid artery and 2 hrs hypoxemia exposure (8% O2 at 37 degrees C). HBA and HBO treatment was administered (2 ATA, once daily for 7 days) in the HBA and HBO groups respectively 1 hr after HIBD. BrdU immunohistochemistry was used to detect the NSCs in the sub-ventricle zone (SVZ) of the lateral ventricle and the dentate gyrus (DG) of the hippocampus. The myelin damage was assessed by myelin basic protein (MBP) immunostaining.

RESULTSThe BrdU-positive cells in the SVZ and the DG of the ischemic hemisphere in the HIBD group were dramatically decreased compared with those of the Normal control group at 3 weeks post-HIBD (P < 0.01). The HBO treatment resulted in an increase of BrdU-positive cells in the DG from 153.7 +/- 37.0 to 193.7 +/- 38.8 (P < 0.05). The nestin expression in the HIBD and HBA groups was reduced compared with that in the Normal control group. There was no difference in the nestin expression between the HBO and the Normal control groups. Hypoxia-ischemia (HI) led to marked myelin damage at 1 week post-HIBD. HBO or HBA treatment alleviated the damage.

CONCLUSIONSThe HBO treatment can result in the proliferation of BrdU-positive cells and alleviate the myelin damage following HIBD in neonatal rats, thereby offering neuroprotectivity against HI insults.

Animals ; Animals, Newborn ; Bromodeoxyuridine ; metabolism ; Female ; Hyperbaric Oxygenation ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; therapy ; Immunohistochemistry ; Intermediate Filament Proteins ; analysis ; Male ; Mice ; Myelin Basic Protein ; analysis ; Nerve Tissue Proteins ; analysis ; Nestin ; Neurons ; cytology ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology