Clinical Study of Neurodegenerative Treatment on Glaucoma by Insulin Intravitreal Injection

Purpose: Our trail was to study insulin intravitreal injection’s (IIV) efficacity and safety to treat glaucoma neurodegeneration.Methods: Eleven subjects (11eyes) were recruited;10 patients treated in a double masked randomized sham controlled including 5 patients received one IIV injection 3UI and 5 patients received one injection of balanced salt solution(BSS) 3UI. A follow up during 168days was realized using Optical Coherence Tomography(OCT) and visual field(VF).The eleventh patient received two IIV injection without masking the injection content within one month between each injection and followed up for 877days.All the patients have a correct ocular pressure and no ocular treatment was stopped.Results: The 5 patients who received IIV revealed a swift improvement of decibel (DB), and remains stable during the first month. The average improvement was 6.62DB during 168days.The 5 patients treated with one BSS injection showed no significant improvement regaining 1. 45DB.The last patient who received two injections showed increase from 7.54DB to 17.22DB with a functional amelioration of 9. 68DB.The OCT examination showed a structural improvement during the first month, then returned to the initial value. No complication was observed during and after the treatment.Conclusion: Insulin shows not only efficacity and safety but more than that, the visual field(VF) of the patients became stable and show no deterioration in all the follow up, which confirmed that insulin act to improve the function rather than structure. it means insulin reconnect the stoma and improve the neurite outgrowth This treatment will change the evolution of this pathology and protect the glaucomatous patients against blindness.

Neuronal differentiation of rat bone marrow mesenchymal stem cells vialentivirus-mediated bone morphogenetic protein 7 transfection / 中国组织工程研究

BACKGROUND: Bone marrow mesenchymal stem cells have the potential to differentiate into neuron-like cells, which have been listed as the preferred stem cells for the treatment of spinal cord injury. However, due to their low differentiation efficiency, it is particularly important to find a factor with high induction ability. Based on literature review and our previous studies, it is speculated that bone morphogenetic protein 7 (BMP-7) gene may play a vital role in promoting the differentiation of bone marrow mesenchymal stem cells into neuron-like cells. OBJECTIVE: To investigate the differentiation of rat bone marrow mesenchymal stem cells into neurons induced by BMP-7 lentivirus vector transfection. METHODS: Bone marrow mesenchymal stem cells of Sprague-Dawley rats were cultured by whole bone marrow adherence method, and then were transfected with LV-GFP when multiplicities of infection were 50, 25, 10, and 1. Green fluorescent protein expression was observed using fluorescence inversion microscope in each group at 3 days after transfection, to confirm the best multiplicity of infection. Passage 3 bone marrow mesenchymal stem cells were divided into blank control group (routine culture), LV-GFP group, and LV-BMP-7-GFP group, followed by transfection at the best multiplicity of infection. After 24, 48, 72, 96, and 120 hours of transfection, MTT assay was used to detect cell survival rate in each group. Immunocytochemical assay was used to detect the expression of nerve cell markers (neurofilament protein 200, synaptophysin-1) after 3 days of transfection. RESULTS AND CONCLUSION: (1) After 3 days of LV-GFP transfection, GFP-positive cells were observed under fluorescence microscopy when multiplicities of infection were 10, 25, and 50, whereas no GFP-positive cells were found when the multiplicity of infection was 1. The average fluorescence intensity was the highest when the multiplicity of infection was 10 (P < 0.05), indicating that multiplicity of infection=10 had the best infection effect. (2) Immunocytochemical results showed that the expression of neurofilament-200 and synaptophysin-1 was negative in the blank control group and LV-GFP group, but positive in the LV-BMP-7-GFP group. The cell body and axon in the LV-BMP-7-GFP group were dyed bright brown. In summary, lentivirus-mediated BMP-7 transfection can promote the differentiation of rat bone marrow mesenchymal stem cells into neuron-like cells.

Activation of PPARγ pathway enhances cellular anti-oxidant capacity to protect long-term cultured primary rat neural cells from apoptosis / 南方医科大学学报

OBJECTIVE@#To study the protective effect of enhanced peroxisome proliferator activated receptor γ (PPARγ) pathway against apoptosis of long-term cultured primary nerve cells.@*METHODS@#A natural aging model was established in primary rat nerve cells by long-term culture for 22 days. The cells were divided into control group, 0.1, 1.0, 5.0, and 10 μmol/L GW9662 intervention groups, and 0.1, 1.0, 5.0, and 10 μmol/L pioglitazone intervention groups. The cell viability was assessed using MTT assay and the cell morphological changes were observed after the treatments to determine the optimal concentrations of GW9662 and pioglitazone. Double immunofluorescence labeling and flow cytometry were used to observe the changes in the number of viable cells and cell apoptosis following the treatments; immunocytochemical staining was used to assess the changes in the anti-oxidation ability of the treated cells.@*RESULTS@#The optimal concentrations of GW9662 and pioglitazone determined based on the cell viability and morphological changes were both 1 μmol/L. Compared with the control group, GW9662 treatment significantly lowered while pioglitazone significantly increased the total cell number and nerve cell counts ( < 0.05), and nerve cells in the cell cultures maintained a constant ratio at about 80% in all the groups ( > 0.05). GW9662 significantly enhanced while pioglitazone significantly lowered the cell apoptosis rates compared with the control group ( < 0.05). GW9662 obviously lowered SOD activity and GSH content in G group ( < 0.05) and increased MDA content in the cells ( < 0.05), and pioglitazone resulted in reverse changes in SOD, GSH and MDA contents in the cells ( < 0.05).@*CONCLUSIONS@#Activation of PPARγ pathway protects long-term cultured primary nerve cells by enhancing cellular anti-oxidant capacity and reducing cell apoptosis, suggesting a potential strategy for anti-aging treatment of the nervous system through intervention of the PPARγ pathway.

Role of basic fibroblast growth factor in epidermal stem cells differentiating into neural stem cells in rats / 中华创伤杂志

Objective To investigate the effect of basic fibroblast growth factor (bFGF) on the differentiation of epidermal stem cells(ESCs) into nerve cells in rats.Methods The epidermal basal layer tissue of newborn SD rats (1-3 days) were isolated and obtained.ESCs were digested and isolated by rapid attachment to a substrate,and the morphology of ESCs was observed under an inverted microscope.ESCs were cuhured with Keratinocytes serum-free medium (K-SFM).The ESCs were grouped and treated according to the density including Group A:0.1 × 107/ml,Group B:0.3 × 107/ml.Group C:0.5 × 1 07/ml,Group D:0.1 × 106/ml,and each group was added bFGF (20 ng/ml).The changes of cell morphology were observed and counted for seven days.The changes of cell markers Nestin and NSE were detected by immunohistochenistry.Results The ESCs of SD rat were isolated successfully.After bFGF induction,the numbers of cells with morphological changes in Groups A and B were larger than those in other two groups in the first 6 days (P ＜ 0.05),and the number in Group A was the largest on the seventh day (P ＜ 0.05).On the third day,Group C had the largest number of cells with changes based on the comparison within the group.There was no change in cell morphology in Group D.Immunohistochemistry showed positive Nestin and NSE.Conclusion The bFGF helps induce the differentiation of ESCs into nerve cells,which is associated with the cell density.

Down-regulated miR-448 relieves spinal cord ischemia/reperfusion injury by up-regulating SIRT1

MicroRNAs play a crucial role in the progression of spinal cord ischemia/reperfusion injury (SCII). The role of miR-448 and SIRT1 in SCII was investigated in this study, to provide further insights into prevention and improvement of this disorder. In this study, expressions of miR-448 and SIRT1 protein were determined by qRT-PCR and western blot, respectively. Flow cytometry was used to analyze cell apoptosis. The endogenous expression of genes was modulated by recombinant plasmids and cell transfection. Dual-luciferase reporter assay was performed to determine the interaction between miR-448 and SIRT1. The Basso, Beattie, and Bresnahan score was used to measure the hind-limb function of rat. The spinal cord ischemia reperfusion injury model of adult rats was developed by abdominal aorta clamping, and the nerve function evaluation was completed by motor deficit index score. In SCII tissues and cells treated with hypoxia, miR-448 was up-regulated while SIRT1 was down-regulated. Hypoxia treatment reduced the expression of SIRT1 through up-regulating miR-448 in nerve cells. Up-regulation of miR-448 induced by hypoxia promoted apoptosis of nerve cells through down-regulating SIRT1. Down-regulated miR-448 improved neurological function and hind-limb motor function of rats with SCII by up-regulating SIRT1. Down-regulated miR-448 inhibited apoptosis of nerve cells and improved neurological function by up-regulating SIRT1, which contributes to relieving SCII.

Protective effect of paeoniflorin on nerve cells in APP/PS1 mice and its mechanism / 中国病理生理杂志

AIM:To investigate the therapeutic and preventive effects of paeoniflorin ( PF) on APP/PS1 mice, and to explore the possible mechanism. METHODS:Fifteen male 5-month-old APP/PS1 non-dominant mice were chosen as normal control group, 15 male 5-month-old APP/PS1 double transgenic mice were used as model group, and 15 male 5-month-old APP/PS1 double transgenic mice treated with 5 mg/kg PF by intraperitoneal injection were allocated in administation group. The learning and memory ability of the mice in each group was detected by Morris water maze. The apoptosis was assessed by TUNEL fluorescence staining. The protein expression of PI3K, Akt, p-PI3K, p-Akt, caspase-3, caspase-9, Bcl-2 and Bax in cerebral cortex and hippocampus was detected by Western Blot. The protein expression levels and distribution of caspase-3 and caspase-9 were detected by immunohistochemistry. RESULTS:(1) Compared with nor-mal control group, the learning and memory ability declined in APP/PS1 model group. Compared with APP/PS1 model group, PF obviously improve the ability of learning and memory in mice. (2) Compared with normal control group, the ap-optosis of nerve cells in APP/PS1 model group significantly increased and distributed in wider areas, while that in PF group was reduced (P<0.05). (3) Compared with APP/PS1 model group, PF could significantly lower pro-apoptotic factors, caspase-3, caspase-9 and Bax (P<0.05), and increase the expression of anti-apoptotic factors, p-PI3K, p-Akt and Bcl-2 (P<0.05). CONCLUSION:PF can up-regulate the expression of Bcl-2 and down-regulate the expression levels of caspase-9, caspase-3 and Bax via the activation of PI3K/Akt pathway, thereby inhibiting the nerve cell apoptosis and pro-tecting the nerve cells, so as to treat neurodegenerative diseases.

The effects of ethanol on the hippocampal neural tissue development and kainite receptor expression in young mouse / 中国神经精神疾病杂志

Objectives To investigate the effects of ethanol on neural development and kainate receptor expression in young mice. Methods Fetal alcohol spectrum disorder model was established by administration of 20% ethanol solu?tion to 7-day-old Kunming mice and control animals received physiological saline (The number of treatment and control were 80 and 40, respectively ). Body weight and general biological features were observed every day. Morris water maze was used to test learning and memory ability. Fluoro-Jade B was used to examine neural cells 24 hours after treatment in additional thirty 7-day-old Kunming mice which were further divided into two groups:a treatment group receiving 20%ethanol solution (n=15) and a control group receiving physiological saline (n=15). The development of neural cells and expression levels of kainite receptors were examined by using immunofluorescence staining. Results The body weight was significantly lighter in treatment group than in control group(control:21.13 ± 1.72g,treatment:13.96 ± 2.98g,P<0.05). Morris test showed that model group had longer latency than control group to find hidden platform(control:21.05± 5.31s,treatment:34.15±3.26s,P<0.05). Spatial probe test revealed that the number of passing through the platform were significantly smaller in model group than in control group(control:2.70 ± 1.25 times,treatment:0.93 ± 0.80 times,P<0.05). Astrocyte development anomaly was evident after ethanol treatment for 7 days. The expression levels of kainite re?ceptor GluR-6 and KA2 were up-regulated in the CA region of the hippocampus after ethanol treatment for 7 days. Con?clusion Kainite receptor GluR-6 and KA2 in CA region of the hippocampus may contribute to ethanol-induced hippo?campal neural development anomaly.

Estrogen receptor ERα36 gene silencing impacts the expression of growth-associated protein in PC12 cells / 中华行为医学与脑科学杂志

Objective To investigate the effects of ERα36 (a novel subtype of estrogen receptor alpha) on growth and proliferation in PC12 cells via examining the expression of growth-associated protein in differentiated PC12 cells after ERα36 gene silencing.Methods Transfection of ERα36-shRNA plasmid into PC12 cells was performed to establish the ERoα36 gene silencing cells model (PC12-36L1,PC12-36L2).Immunocytofluorescence was used to examine the expression of ERα36,and Western blot was used to analyze the expression of PCNA,cyclinD1 and MAPK in the PC12 cells.Results ① ERα36 was expressed in both cell types(PC12-36C1,PC12-36L1 and PC12-36L2).Compared with PC12-36C1,PC12-36L2 cells(OD value were respectively 0.95±0.05,0.78±0.10),PC12-36L1 cells significantly decreased expression of ERα36(OD value 0.47±0.12,P＜0.01).② Compared with PC12 and PC12-36C1 cells,PC12-36L1 cells were significantly higher expression of PCNA,CyclinD1 and p-MAPK(P＜0.01)(OD value of PCNA,CyclinD1 and p-MAPK:PC12 cells were respectively 1.00±0.05,1.00± ±0.11,1.00±0.05,PC12-36C1 cells were respectively 1.09±0.15,0.92±0.23,1.12± 0.08,PC12-36L1 cells were respectively 1.74±0.12,2.20±0.25,1.77±0.06).Conclusion ERα36 gene silencing can promote the growth and proliferation in PC12 cells.It suggests that the lower expression of ERα36 may be related to the diseases in nervous system such as brain tumor.

Basic Fibroblast Growth Factor-chitosan Carriers Induce Bone Marrow-Derived Mesenchymal Stem Cells to Differentiate into Nerve Cells / 中国康复理论与实践

@#ObjectiveTo explore the effect of bFGF-chitosan carriers on inducing bone marrow-derived mesenchymal stem cells (MSCs) to differentiate into nerve cells.MethodsMSCs were detected by immunohistochemistry and Western blot after they were induced by bFGF-chitosan carriers to differentiate into neurons. The MTT chromometry assay was carried out to determine cell viability.ResultsThe proportion of express neural stem cells marker Nestin, and neuronal markers class Ⅲ β-tubulin and MAP-2 was 83.54% after MSCs induced by bFGF-chitosan carriers.ConclusionbFGF-chitosan carriers can induce MSCs to differentiate into nerve cells with a high percentage.

Neuroprotective effects of erythropoietin on cultured retinal neurocytes by glutamate / 上海第二医科大学学报

Objective To investigate whether erythropoietin(Epo) is potentially beneficial in protecting cultured retinal neurocytes.Methods Primary isolated retinal nerve cells were cultured.Expressions of Epo and EpoR protein in cultured retinal neurocytes were decected by immunohistochemical analysis.Survival of cultured neurocytes that were incubated in the presence of Epo or glutamate in the presence or absence of Epo were estimated by determining the activity of their mitochondrial dehydrogenases using the MTT assay.Results Epo and EpoR protein were expressed on the cultured retinal neurocytes.The presence of different concentrations of Epo did not improve the survival of retinal neurocytes,and Epo could prevent glutamateinduced toxicity. Conclusion Epo is beneficial in protecting mixed cultured retinal neurocytes from glutamate-induced cytotoxicity.

Effect of 8-Br-cAMP and IPP on the expression of tau protein in various neural cell lines / 中华老年医学杂志

ObjectiveTo study the effect of 8-Br-cAMP and isopentenyl pyrophosphate (IPP) on the ex pression tau protein in various neural cell lines at the level of signal transdu ction. MethodsNG108-15, PC12 and SH-SY5Y cells were treated with 8-Br-cAMP and IPP in the 37?C incubator for 30 min, then the tau-enriched samples were analyzed by West ern blot. ResultsSH-SY5Y cells only expressed the isoform of tau protein with the lowest molecul ar weight; PC12 cells mainly expressed high molecular weight tau, and several ot her isoforms of tau; NG108-15 expressed a little of isoform of tau. In the expe rimental conditions, the expression of tau was altered with 8-Br-cAMP and IPP in NG108-15, SH-SY5Y and PC12 cells. Conclusions8-Br-cAMP and IPP does not alter the expression of tau in NG108-15, SH-SY5Y and PC12 cells.

THE EFFECTS OF DIFFERENT INDUCERS ON THE DIFFERENTIATION OF NONHEMATOPOIETIC STEM CELLS INTO NERVE CELLS IN HUMAN UMBILICAL CORD BLOOD (HUCB) / 解剖学报

GFAP.Conclusion RA is the best factor for neurons and astroglia,and RA+EGF+bFGF are the best for oligodendrocytes.

ULTRASTRUCTURAL STUDY OF DISSOCIATED CHICKEN EMBRYO BRAIN NERVE CELLS CULTURED IN VITRO / 解剖学报

The ultrastructure of dissociated brain nerve cells from embryonic chicken cult ivated in vitro was studied both under SEM and TEM. Primitive synaptic junctions among the bipolar and multipolar neuronal cell bodies and its processes forming networks of fibers appeared after 4 days of culture. The presynaptic bags and desmosome-like cell junctions were observed simutaneously. Axo-dendritic, axo-somatic and axo-axonal synapses increased in number after 7 days of incubation onwards. Neuronal degeneration and proliferation of fibrous glial cells were evident in10 and 14 days cultures.