The mechanism of neuronal injury and repair after focal cerebral ischemia

AIM:To explore the mechanism of neuronal injury and repair by investigating the expression of caspase-3 and apurinic/apyrimidinic endonuclease (APE/Ref-1) after focal cerebral ischemia. METHODS:A model of middle cerebral artery occlusion in rats was performed. The expression of caspase-3P₂₀ and APE/Ref-1 was examined by immunohistochemistry staining, TUNEL was applied to detected DNA damage, and double labeling with TUNEL and APE/Ref-1 was used to determine the relationship between APE/Ref-1 and DNA damage. RESULTS:The active subunit P₂₀ of caspase-3 was predominantly expressed within ischemic penumbra. The peak time of caspase-3P₂₀ positive cells preceded the appearance of TUNEL. With aggravation of cerebral ischemia, APE/Ref-1 immunoreactive cells in penumbra were significantly decreased. CONCLUSION:The activation of caspase enzymatic cascade following cerebral ischemia leads to degradation in DNA, meanwhile, decrease in DNA repair molecules or the failure of DNA repair may deteriorate the course.     

Sca-1+ cells from mouse fetal liver can differentiate into neurons in vitro

AIM: To study whether Sca-1⁺ cells from fetal liver can be induced to differentiate into neuronal cells in vitro. METHODS:Sca-1⁺cells from 14 5-days-old murine fetal liver were isolated with a magnetic cell sorting kit, and were cultured in Dulbecco s modif ied Eagle s medium(DMEM)/F12 supplemented with 10%fetal bovine serum(FBS), and passaged at a rat io of 1 3 when cells reached more than 80%confluence.The 5 passage cells were induced by 10^-3mol/Lβ-mercaptoethanol(β-ME)and 5×10^-7 mol/L all-trans-retinoic acid(RA)for 24 hours, and then incubated in serum-free medium for 5 hours to 5 days.The characteristics of treated cel s were assayed by immunocytochemistry staining analysis at 5 hours, or 5 days.RESULTS: Cells treated with β-ME and RA exhibited neuronal phenotype and expressed neuron-specific protein such as neuron-specific nuclear protein (NeuN), neuronfilament-M, and neuron-specific tubulin-1 (TuJ-1) but not tau, MAP-2, or the astrocyte-specific marker glial fibrillary acidic protein (GFAP).CONCLUSION: Sca-1⁺ cells from fetal liver, of which most are regarded as hematopoietic stem cells, could differentiate into early immature neuronal cells in vitro. These findings suggest that Sca-1⁺ cells from fetal liver may be an alternative source in cell therapy and gene therapy of neural dysfunction.     

Adult rat and human bone marrow mesenchymal stem cells differentiate into neurons with Musk's polypeptide

AIM: To investigate the differentiation from adult rat and human bone marrow mesenchymal stem cells (BMMSCs) into neuron with musk polypeptide (Mu-P).METHODS: Adult rat and human BMMSCs were induced with Mu-P.Neuron-specific enolase (NSE),neurofilament (NF),Nestin,glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry.RESULTS: Simple methods with Mu-P induced adult rat and human BMMSCs exhibiting a neuronal phenotype,expressing Nestin at 3 hours to 5 hours,and expressing NE and NF at 5 hours to 7 days.But the neuron-like cells didn't express the glial astrocyte marker GFAP.CONCLUSION: Adult rat and human BMMSCs can be induced to differentiate into neurons with Mu-P.     

Advances in studying the differentiation into neurons of mesenchymal stem cells and their application

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can proliferate and differentiate into marrow and non-marrow cell types, such as adipocytes, chondrocytes, myocytes, and so on. In recent years, many researchers have studied whether MSCs are capable of differentiation into neurons in vivo and ex vivo. The result that MSCs-derived neurons express NSE and NF, but don't express GFAP suggests MSCs can differentiate into neurons, some researchers have achieved success in promoting functional recovery in Pakinsons and transactional spinal cord injury rat models by use of MSCs-derived neurons. Therefore, MSCs-derived neurons will play an important role in the therapy for a variety of diseases of the nervous system.     

Resveratrol protects cortical neurons in infant rats with bacterial meningitis through miR-223-3p/NLRP3 pathway

AIM To investigate the protective effect of resveratrol (Res) on cortical neurons in rat bacterial meningitis (BM) model. METHODS Group B hemolytic Streptococcus was injected via the posterior cistern to establish a BM model. Resveratrol was administered intranasally and microRNA-223-3p (miR-223-3p) antagomir was administered by intracerebroventricular injection. HE staining was used to observe the pathological changes of the brain tissue. Loeffler scoring method was used to evaluate the neurobehavioral functions. TUNEL staining was used to detect neuronal apoptosis. The expression of interleukin-1β (IL-1β), IL-18, glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (Iba1) was detected by immunofluorescence staining. The protein levels of nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3）, cleaved caspase-1, IL-1β and IL-18 were determined by Western blot. The expression level of miR-223-3p was detected by RT-qPCR. Online software TargetScan was used to search for the complementary nucleotide sequences between miR-223-3p and NLRP3 mRNA. RESULTS Compared with sham group, the thickness of meninges in BM model was increased, the neurological score was decreased (P<0.05), and the number of TUNEL positive neurons was increased significantly (P<0.05). Astrocytes and microglia were activated, the fluorescence intensity of IL-1β and IL-18 was increased (P<0.05), and the expression levels of NLRP3, cleaved caspase-1, IL-1β, IL-18 and miR-223-3p were increased (P<0.05). Compared with BM group, after treatment with resveratrol, the neurological score was increased (P<0.05), the number of TUNEL positive neurons was decreased significantly (P<0.05), and the inflammatory response of astrocytes and microglia was suppressed. The fluorescence intensity of IL-1β and IL-18 was decreased (P<0.05), the protein levels of NLRP3, cleaved caspase-1, IL-1β and IL-18 were decreased (P<0.05), and the expression level of miR-223-3p was increased (P<0.05). A nucleotide sequence in the 3'-UTR of NLRP3 mRNA might be targeted by miR-223-3p. In the brain of rat BM model, compared with antagomir control group, the expression of NLRP3 was increased in miR-223-3p antagomir group with resveratrol treatment (P<0.05). CONCLUSION Resveratrol may reduce the inflammatory death of cortical neurons in BM model of infant rats through miR-223-3p/NLRP3 pathway, thus playing a protective role for the neurons.     

Shikonin inhibits neuronal apoptosis induced by OGD through targeting PI3K/Akt signaling pathway

AIM: To explore the effect of shikonin on rat primary cortical neurons in oxygen-glucose deprivation (OGD)-induced injury model.METHODS: The neurons were pretreated with shikonin at different concentrations (0.02, 0.2, 2 and 20 μmol/L) followed by treatment with OGD. Lactate dehydrogenase (LDH) release assay and fluorescein diacetate/propidium iodide (FDA/PI) double staining were used to detect neuronal viability and apoptosis, and then the optimal concentration of shikonin was determined. LY294002 (PI3K/Akt signaling pathway inhibitor, 1 μmol/L) was added before the addition of shikonin, and the protein level of p-Akt (Ser473) in the neurons was determined by Wes-tern blot. LDH release assay and FDA/PI double staining were also used to detect neuronal viability and apoptosis.RESULTS: A certain concentration (0.2~20 μmol/L) of shikonin increased the viability of impaired neurons (P<0.05) and the protein level of p-Akt (Ser473) in the neurons (P<0.05). The effect of shikonin on neuronal p-Akt (Ser473) levels and the cell death were blocked by LY294002 (P<0.05).CONCLUSION: A certain concentration of shikonin reduces OGD-induced apoptosis of rat primary cortical neurons by activating PI3K/Akt signaling pathway.     

Screening differentially expressed genes involved in apoptosis of primary cultured rat cerebellar granule neurons by mRNA differential display RT-PCR

AIM: To Screen and identify differentially expressed genes that involved in apoptosis model in rat cerebellar granule neurons (CGNs).METHODS: The rat cerebellar granule neurons were isolated and primarily cultured.Fluorescent differential display RT-PCR (FDD RT-PCR) was performed to screen differentially expressed ESTs in the apoptosis model of primarily cultured rat CGNs.ESTs were subcloned into pGEM-T EasyTM vector and then sequenced.Alignment assay in non-redunant database was applied for encoding information.Reverse Northern blotting was used to appraise the results from DDRT-PCR.RESULTS: 164 pieces of differentially expressed ESTs were obtained by FDDRT-PCR.17 of them were subcloned and sequenced.5 ESTs of 17 were confirmed to be positive results by reverse Northern blotting.CONCLUSION: DD-PCR is a rapid,simple-operation and sensitive method for screening differentially expressed genes,which would contribute to the molecular mechanisms of apoptosis/survive of CGNs.     

Effect of SAPK/JNK signal pathway on neuronal apoptosis in cerebral infarction rats

AIM: To investigate the molecular mechanism of neuronal apoptosis by observing the changes of key proteins in SAPK/JNK and Bcl-2/Bax signal pathways after brain infarction. METHODS: The cortical infarction was induced by photochemistry, namely photothrombotic cortical injury (PCI). Thirty-six Sprague-Dawley rats were randomly divided into 2 groups: PCI group and sham-operated group. The ipsilesional cortex was harvested for histomorphometry and transmission electron microscopy 7 days after PCI. Some key proteins including p-JNK1, p-JNK2, p-c-Jun, p-ATF-2, total JNK1, total JNK2, Bcl-2 and Bax were detected by Western blotting analysis.RESULTS: The cortical infarction in rats was successfully induced by photochemistry. The apoptosis of neurons in cortex was more obvious in PCI group than that in sham-operated group 7 days after PCI. The levels of p-JNK1, p-JNK2, p-c-Jun and p-ATF-2 in PCI group were significantly higher than those in sham-operated group, whereas the ratio of Bcl-2/Bax was significantly lower(P<0.05). CONCLUSION: Apoptosis is a major contributor to neuronal loss induced by cerebral hypoxia-ischemia for a long period after cortical infarction. The process is related to some apoptotic proteins such as Bcl-2/Bax and the SAPK/JNK signal pathways activated by ischemic injury.     

Influence of mitochondrial deficiency on expression of microtubule-associated proteins in primary cultured hippocampal neurons of neonatal rats

AIM: In order to study the relationship between mitochondrial deficiency and Alzheimer's disease(AD), we used sodium azide, a specific inhibitor of cytochrome C oxidase (COX), to develop a cell model of mitochondrial complex IV deficiency and investigated the impairment of microtubules and microtubule-associated proteins. METHODS: Primary cultured hippocampal neurons of hewborn rats were exposed to sodium azidethen cell viability was measured by MTT method; cell morphology, immunofluorecence-stained cellular microtubules and microtubule-associated proteins were observed by confocal microscopy. RESULTS: Primary cultured hippocampal neurons were exposed to 8-128 mmol/L sodium azide for 3-24 h, MTT absorbance decreased dose-and time-dependently. Exposed to 64 mmol/L sodium azide for 6 h, the processes of cells retracted, synapses disappeared, axons were shortened under contrast microscope. Meanwhile, microtubles were disassembled and became disorderly, the expression of microtubule-associated proteins were also reduced especially in the processes observed by confocal microscopy. CONCLUSION: Sodium azide inhibits the assembly and polymerization of tubulin in microtubules which may be reduced by low expression of microtubule-associated proteins in nerve cells. The damage of axons induced by microtubule collapse further blocks the intercellular signal transduction and intracellular material transportation which are important causes in cell death.     

Protective effect of progesterone on PC12 cells injured by oxygen-glucose deprivation

AIM: To investigate the effects of progesterone on the cell viability and expression of glucose transporter type 3(GLUT3) in PC12 cells injured by oxygen-glucose deprivation (OGD) in attempt to prove the neuroprotection of progesterone (PROG) against the hypoxic-ischemic injury in cultured cells in vitro. METHODS: Well-differentiated PC12 cells induced by nerve growth factor were randomly divided into 3 groups. In normal group, the cells were cultured without OGD treatment. In OGD group, the culture medium was replaced by glucose-free medium and the cells were transferred to a humidified incubation chamber flushed by a gas mixture of 95% N₂ and 5% CO₂ for 30 min. After that, the cells were fed with glucose-supplemented medium and cultured under normoxic condition for 24 h. In PROG+OGD group, the cells were given the same treatments as those in OGD group except that the medium contained progesterone at concentration of 10 nmol/L. Cellular morphological changes were observed after OGD for 30 min. The cell viability was assessed by WST-8 assay. The degree of the cell damage was evaluated by determining lactate dehydrogenase (LDH) leakage. The expression of GLUT3 at mRNA and protein levels was examined by RT-PCR and Western blotting, respectively. RESULTS: Progesterone attenuated the cellular swelling, decreased the leakage of LDH and improved the viability of PC12 cells injured by OGD (P<0.01). The expression of GLUT3 at mRNA and protein levels in PC12 cells in PROG+OGD group was significantly higher than that in OGD group (P<0.05). CONCLUSION: Progesterone has protective effect on in vitro cultured PC12 cells injured by OGD. The mechanism may be related to the up-regulation of GLUT3 protein.