Effect of electroacupuncture on microglia phenotype in injured abducens nerve

AIM: To investigate the effect of electroacupuncture treatment on microglia phenotype in Beagle dogs with abducens nerve injury. METHODS: Healthy adult Beagle dogs were randomly divided into 4 groups:normal control group, sham operation group, injury group and electroacupuncture treatment group, with 5 dogs in each group. In sham operation group, only the isolated nerves were exposed. In injury group, the dogs were restrained when electroacupuncture was performed in electroacupuncture treatment group. In electroacupuncture treatment group, electroacupuncture was performed on the base of dog nerve injury. The experiment was continued for 2 weeks. Western blot was used to detect the expression levels of M1 and M2 markers in the microglia. RESULTS: The expression of inducible nitric oxide synthase (iNOS) in injury group was significantly higher than that in control group (P<0.05). In electroacupuncture treatment group, the expression of iNOS was significantly lower than that in injury group (P<0.05). The expression of interleukin-1β (IL-1β) in injury group and electroacupuncture treatment group was higher than that in control group and that in electroacupuncture treatment group was significantly lower than that in injury group (P<0.05). The expression of arginase in injury group and electroacupuncture treatment group was higher than that in control group (P<0.05), and that in electroacupuncture treatment group was higher than that in injury group (P<0.05). Meanwhile, the expression of brain-derived neurotrophic factor (BDNF) in injury group and electroacupuncture treatment group was increased compared with control group (P<0.05). But the expression of BDNF in electroacupuncture treatment group was higher than that in injury group (P<0.05).CONCLUSION: After abducens nerve injury, electroacupuncture treatment reduces the expression levels of M1 marker proteins in microglia and increases the expression levels of M2 marker proteins.     

CX3CR1 mediates the neuroprotective effect of triptolide on 1-methyl-4-phenylpyridinium-induced hemiparkinson rats

AIM: To investigate the effect of triptolide on the inhibition of microglial activation in 1-methyl-4-phenyl pyridinium (MPP⁺)-induced hemiparkinson disease rats.METHODS: The rat model of Parkinson disease was established by intranigral injection of MPP⁺. The rats were randomly divided into sham group, MPP⁺ group, triptolide group and vehicle group. The survival of dopaminergic neurons was detected by the immunofluorescence of tyrosine hydroxylase (TH) in the substantia nigra (SN). The activation of microglia was determined by immunofluorescence of OX-42 (microglia marker) in the SN. The expression of chemokine receptor CX3CR1 in SN was measured by Western blotting.RESULTS: Intranigral injection of MPP⁺ increased the fluorescence intensity of the microglial marker, and promoted DA neuron degenerative death. Immunohistological analysis showed that the OX-42 density was decreased (P<0.01) and tyrosine hydroxylase (TH) positive neurons were increased in the triptolide group (P<0.01). The expression of CX3CR1 was lower in triptolide group than that in model group (P<0.05).CONCLUSION: Triptolide may improve PA neurons function in MPP⁺-induced rats through inhibiting CX3CR1 expression and microglial activation.     

Studies on the isolation,purification,specific molecule expression and phagocytosis of human microglia

AIM: To explore a method of isolation, purification, culture and identification of human microglia. METHODS: The brain tissue from abortive fetus was sterilely obtained, then chopped and triturated gently. The suspension was filtered and centrifuged to separate and isolate mixed glia cells. The cell density was adjusted to 2×1010 cells/L with DMEM/F12 complete medium and cultured in 75 cm2 flask at 37 ℃ in CO2 incubator (marked as first 0 d). After 7-10 days culture, floating cells including microglia and oligodendrocytes appeared in the flask. The first time purification was carried out to obtain highly purified microglia, for which the flasks were shaken gently. The floating cells were collected and transferred into a new flask (marked as second 0 d). 4-5 days later, when microglia and oligodendrocytes grew in confluent state, the second purification was performed, for which the mixed cultured cells were digested with trypsin-EDTA and cell suspension was centrifuged, then the cell pellet was suspended by DMEM/F12 complete medium and cultured in flask (marked as 0 h). After 2 h, non-microglial cells including oligodendrocytes and cell debris were removed, and fresh medium was added into the adherent cells. In this way, the whole procedure of microglia purification was completed and the highly purified microglial cells were obtained. After purification, the expression rate of CD45, CD11b and CD68 on/in microglia were detected by laser-confocal microscopy and flow cytometry on the basis of immuno-fluorescence staining to identify the purity of microglial cells. Microglial phagocytotic function was evaluated by phagocytosis of fluorescent microspheres. The level of activation was identified by the phenomena of membrane ruffling in combination with fluorescent phalloidin staining. RESULTS: More than 98% of the microglias that we isolated and purified expressed CD45,CD11b and CD68. Almost all of the microglias could phagocytize fluorescent microbeads CONCLUSION: We succeed in isolating and purifying human microglias,which express CD45,CD11b and CD68.Almost all of the cells exhibit phagocytic function.So these cells will be useful for further functional and proteomic studies.     

Inhibition of Toll-like receptor 9 activation in microglia after oxygen-glucose deprivation and reoxygenation protects neurons from damage

AIM: To observe the Toll-like receptor 9 (TLR9) activation in microglia BV-2 cells after oxygen-glucose deprivation and reoxygenation (OGDR), and its effects on neuronal apoptosis. METHODS: The BV-2 cell supernatants were collected after the corresponding treatment and added to mouse primary cortical neurons after OGDR for 4 h, followed by normal culture for 24 h. The cells were divided into normal BV-2 group, NC-siRNA group, TLR9-siRNA group, OGDR group, OGDR+NC-siRNA group, OGDR+TLR9-siRNA group and control group (without adding BV-2 cell supernatant). The changes of the neuronal morphology were observed under an inverted phase- contrast microscope, and the neuronal apoptosis was detected by TUNEL. The protein expression of cleaved caspase-3 was detected by Western blotting. RESULTS: After OGDR, the axon turned thin, twisted and broken, and neuronal swelling, decrease in refraction and vacuolar degeneration were observed. The green-stained apoptotic bodies in the neurons in all groups were positive. Compared with control group, the caspase-3 protein levels in other groups were increased. Compared with the normal BV-2 group, the caspase-3 protein in OGDR group and TLR9-siRNA group was increased. Compared with OGDR+TLR9-siRNA group, the caspase-3 protein in TLR9-siRNA group and OGDR group was decreased. CONCLUSION: After OGDR, TLR9 activation in BV-2 cells induces neuronal apoptosis with the increase in caspase-3 protein level. Inhibition of TLR9 expression reduces neuronal damage.     

α7nAChR is involved in anti-inflammation of physiological concentrations of glucocorticoids

AIM: To explore the role of α7 nicotinic acetylcholine receptor (α7nAChR) in anti-inflammation of glucocorticoids (GCs) at physiological concentrations. METHODS: MTT assay was used to measure the viability of BV-2 cells, which were processed by hydrocortisone at different concentrations. On the basis of inflammatory model induced by LPS in BV-2 cells, experimental groups were divided as follows: (1) control; (2) LPS; (3) GCs＋LPS; (4) methyllycaconitine (MLA)＋GCs＋LPS. The levels of TNF-α and IL-1β in the cell supernatants were detected by ELISA. RESULTS: Hydrocortisone at concentrations of 2 000 and 1 000 nmol/L decreased the cell viability to (76.9±5.5)% and (90.8±7.3)%, respectively, indicating the cellular injury by GCs at over-physiological doses. LPS significantly induced the releases of TNF-α and IL-1β in a time- and dose-dependent manner in BV-2 cells. Hydrocortisone at physiological concentrations (500 and 250 nmol/L) reduced the releases of TNF-α and IL-1β in BV-2 cells stimulated by LPS, and MLA at concentration of 10 nmol/L antagonized the anti-inflammatory effect of GCs. CONCLUSION: α7nAChR is involved in the anti-inflammatory effect of the physiological concentrations of GCs.     

Effects of electroacupuncture pretreatment on survival,brain injury and cognitive function in rats after limb ischemia/reperfusion

AIM：To investigate the effects of electroacupuncture (EA) pretreatment on survival, brain injury and cognitive function in rats after limb ischemia/reperfusion (LI/R). METHODS：One hundred and thirty-two healthy male SD rats weighing 255~300 g were randomly divided into 3 groups (n=44 each)：sham operation group (sham), LI/R group and LI/R plus EA pretreatment (IL/R+EA) group. The LI/R model was made by a method that the bilateral femoral arteries were occluded for 3 h with atraumatic microclips followed by 48 h of reperfusion. In sham group, sham operation was performed. The EA pretreatment was conducted twice a day for 14 d prior to the LI/R event. EA pretreatment included the following acupoints：Baihui (GV20), Zusanli (ST36) and Xuehai (SP10). The survival rate within 7 d following LI/R was calculated. The changes of cognitive function were detected 48 h after reperfusion using Morris water maze test. The cerebral water content was determined by detecting the wet and dry weight. Microglial cells were evaluated following immunolabeling of Iba1 (a marker of microglia). The protein level of cleaved caspase-3 in the hippocampus was measured by Western blotting. The neuronal apoptosis was detected using TUNEL method. Meanwhile, the changes of pathological structure in hippocampus were observed under light microscope. The activity of MPO and SOD, and the content of ROS and MDA were also investigated. RESULTS：Compared with sham group, the Iba1 positive cells, the protein level of cleaved caspase-3, the apoptotic index, and the levels of ROS/MDA and MPO activity in LI/R and LI/R+EA groups increased significantly. The normal hippocampal neurons reduced, and SOD activity decreased significantly in hippocampus. The survival rates of the rats within 7 d decreased, the latency and swimming distance increased, and the number of crossing the platform reduced. Compared with LI/R group, the above indexes in LI/R+EA group were markedly improved. CONCLUSION：EA stimulation improves the survival rate and cognitive dysfunction, and reduces brain damage in LI/R rats by preventing microglial activation and attenuating oxidative stress.     

雷公藤内酯醇对AD细胞模型炎性因子的影响

[目的]以Aβ1-42寡聚体为工具药建立AD细胞模型,观察雷公藤内酯醇（T10）对AD的防治作用并初步探讨其作用机制。[方法]用不同浓度的T10（10-11、10-10、10-9、10-8mol/L）预孵育小胶质细胞12h,然后加入10μmol/LAβ1-42共孵育12h,ELISA法检测上清TNF-α、IL-1β的含量,收获条件培养基,加入海马神经元中,作用24h,MTT法检测神经元存活率。[结果]在体外T10可减少Aβ1-42诱导的小胶质细胞TNF-α、IL-1β的释放,提高海马神经元的存活率。[结论]T10可通过抑制小胶质细胞激活保护海马神经元。     

Microtubule associated protein 2 and choline acetyltransferase immunoreactivity in the lumbar spinal cord of young adult and aged dogs

German Shepherds are a good model for research about aging and neurological disorders such as lumbosacral spinal canal stenosis. We compared neurons, glia and cholinergic neurons in the ventral horn of the lumbar spinal cord (L₃) between adult (1–2 years old) and aged (10–12 years old) groups. Any pathological findings were not found by hematoxylin and eosin staining and neurological examination, and the number of NeuN (a marker for neurons)-positive neurons were similar in both groups. Microtubule-associated protein 2 (MAP2) immunoreactive dendrites in the aged dog were decreased without any change in β-tubulin protein level. Glial fibrillary acidic protein (a marker for astrocytes) and ionized calcium-binding adapter molecule 1 (a marker for microglia) immunoreactivity were not significantly changed in both groups. The number of ChAT immunoreactive neurons was decreased; however, its protein level was not significantly changed in the aged group. These results suggest that numbers of ventral horn neurons are not changed, but cholinergic neurons may change in aged dogs compared to adult dogs.     

Emodin reduces hypoglycemia/hypoxia-induced injury of microglia by TLR4/NF-κB signaling pathway

AIM: To investigate the mechanism of emodin on the protection of glucose-deficient/anoxic microglia. METHODS: A microglia BV2 cell model induced by hypoglycemia/hypoxia (HH) was established. The glucose-deficient/anoxic cells treated with emodin were labeled as HH+emodin (20, 40 and 80 μmol/L) groups. The BV2 cells with TLR4 over-expression treated with emodin under hypoglycemia/hypoxia condition was labeled as HH+pcDNA-TLR4+ emodin (40 μmol/L) group. The cell viability was measured by MTT assay. Lactate dehydrogenase (LDH) and tumor necrosis factor-α (TNF-α) levels were detected by ELISA. The apoptosis was analyzed by flow cytometry. The protein levels of Bax, Bcl-2, TLR4, p-IκB and IκB were determined by Western blot. RESULTS: Compared with HH+DMSO group, the viability was significantly increased, the levels of LDH and TNF-α and apoptotic rate were significantly decreased, the protein levels of Bax, TLR4 and p-IκB were significantly decreased, the protein level of Bcl-2 was significantly increased in HH+emodin groups (P<0.05). Over-expression of TLR4 reversed the effect of emodin on promoting the viability and inhibiting apoptosis in the BV2 cells. CONCLUSION: Emodin has a protective effect on hypoglycemia/hypoxia induced microglia, and its mechanism may be related to the inactivation of TLR4/NF-κB signaling pathway.