Transplantation of Pro-Oligodendroblasts, Preconditioned by LPS-Stimulated Microglia, Promotes Recovery After Acute Contusive Spinal Cord Injury.

Spinal cord injury (SCI) is a significant clinical challenge, and to date no effective treatment is available. Oligodendrocyte progenitor cell (OPC) transplantation has been a promising strategy for SCI repair. However, the poor posttransplantation survival and deficiency in differentiation into myelinating oligodendrocytes (OLs) are two major challenges that limit the use of OPCs as donor cells. Here we report the generation of an OL lineage population [i.e., pro-oligodendroblasts (proOLs)] that is relatively more mature than OPCs for transplantation after SCI. We found that proOLs responded to lipopolysaccharide (LPS)-stimulated microglia conditioned medium (L+M) by preserving toll-like receptor 4 (TLR4) expression, improving cell viability, and enhancing the expression of a myelinating OL marker myelin basic protein (MBP), compared to other OL lineage cells exposed to either LPS-stimulated (L+M) or nonstimulated microglia conditioned medium (LM). When L+M-stimulated proOLs were intrathecally delivered through a lumbar puncture after a T10 thoracic contusive SCI, they promoted behavioral recovery, as assessed by the BassoBeattieBresnahan (BBB) locomotor rating scale, stride length, and slips on the grid tests. Histologically, transplantation of L+M proOLs caused a considerable increase in intralesional axon numbers and myelination, and less accumulation of invading macrophages when compared with the vehicle control or OPC transplantation. Thus, transplantation of proOLs, preconditioned by L+M, may offer a better therapeutic potential for SCI than OPCs since the former may have initiated the differentiation process toward OLs prior to transplantation.

Hypoxic preconditioning up-regulates glucose transport activity and glucose transporter (GLUT1 and GLUT3) gene expression after acute anoxic exposure in the cultured rat hippocampal neurons and astrocytes.

Hypoxic preconditioning has been shown to increase the hypoxic tolerance of brain neurons. However, the mechanism underlying the increased hypoxic tolerance has not been well elucidated. Since anaerobic glycolysis is the only pathway for a vertebrate cell to produce energy under anoxic conditions, which needs a large amount of glucose, we hypothesize that glucose transport, the rate-limiting step for glucose metabolism, plays a critical role in the hypoxic tolerance induced by hypoxic preconditioning. In this study, the effects of hypoxic preconditioning on glucose transport activity and the gene expression of two major forms of glucose transporters (GLUT1 and GLUT3) in the brain were investigated in cultured rat hippocampal neurons and astrocytes. The neuronal and astroglial cultures were preconditioned for 6 days by intermittently exposing the cells to sublethal hypoxic gas mixture (1% O2/10% CO2/89% N2) for 20 min each day. 24 h after the last hypoxic exposure, the cells were exposed to a lethal anoxic gas mixture (10% CO2/90% N2) for 6 h and the uptake rate of [3H] 2-deoxyglucose (2-DG) and the levels of GLUT1 and GLUT3 glucose transporter mRNAs in the cells were examined immediately after anoxic exposure. The neurons and astrocytes preconditioned with hypoxia showed higher 2-DG uptake rates than the non-preconditioned cells. Compatible with the change in 2-DG uptake, hypoxic preconditioning also increased GLUT1 mRNA levels in the astrocytes and GLUT1 and GLUT3 mRNA levels in the neurons. The neurons preconditioned by hypoxia displayed increased anoxic tolerance. However, when glucose uptake in the neurons was blocked by cytochalasin B, the anoxic tolerance was almost abolished. These results suggest that glucose transport is critical to neuronal survival during anoxic exposure and the increased glucose transport activity is probably one of the important mechanisms for the enhanced hypoxic tolerance induced by hypoxic preconditioning.

[Effects of ginkgolide B against damage of cultured hippocampal neurons caused by glutamate].

AIM: To investigate protective effects of ginkgolide B (GB) in different administration modes on glutamate-induced neuronal damage. METHODS: Essential GB were obtained by supercritical CO2 fluid extraction. Glutamate excitotoxicity were examined in primary cultures from neonatal Wistar rat, by using of Trypan blue dye staining, testing the lactate dehydrogenase leakage from cultured neurons and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) method. The protective effects of GB in different administration modes (pre-treatment and post-treatment) were adopted and compared with the NMDA receptor uncompetitive antagonist-MK-801 in acute-treatment. RESULTS: Treatment with GB in two administration modes both could increase ratio of surviving neuron, decrease LDH efflux and reduce ratio of neuron apoptosis in different degree, depended on dose in certain range. The protective effect of pre-treatment was superior to post-treatment, but inferior to MK-801. CONCLUSION: GB can protect neurons against glutamate damage, and preventive using has more efficiency. The potential mechanism of its neural protection may be not only related to PAF receptor. If the predominant protection effect of GB in pretreatment is considered, precautionary intervention to high-risk population could have more value.

Neurogenesis in the adult rat brain after intermittent hypoxia.

Intermittent hypoxia has been found to prevent brain injury and to have a protective role in the CNS. To address the possible causes of this phenomenon, we made investigative effort to find out whether intermittent hypoxia affects neurogenesis in the adult rat brain by examining the newly divided cells in the subventricular zone (SVZ) and dentate gyrus (DG). The adult rats were treated with 3000 and 5000 m high altitude 4 h per day for 2 weeks consecutively. 5-Bromo-2-deoxyuridine-5-monophosphate (BrdU) immunocytochemistry demonstrated that the BrdU-labeled cells in the SVZ and DG increased after 3000 and 5000 m intermittent hypoxia. The number of BrdU-labeled cells in the SVZ returned to normal level 4 weeks following intermittent hypoxia. However, the BrdU-labeled cells in the DG had a twofold increase 4 weeks subsequent to intermittent hypoxia. From these data, we conclude that intermittent hypoxia facilitates the proliferation of neural stem cells in situ, and that the newly divided cells in the SVZ and DG react differently to hypoxia. We are convinced by these findings that the proliferation of neural stem cells in SVZ and DG may contribute to adaptive changes following intermittent hypoxia.

Underlying mechanism of hypoxic preconditioning decreasing apoptosis induced by anoxia in cultured hippocampal neurons.

It is known that hypoxic preconditioning (HP, a brief period of sublethal hypoxia) provides neuroprotection against subsequent severe anoxia, but the mechanisms of this increased tolerance have not been fully elucidated. A hypoxic preconditioning model was established by exposing a 4-day hippocampal culture to 1% O(2) for 20 min/day for 8 days. The preconditioning significantly decreased the number of apoptotic neurons at reoxygenation 24 h after 4 h of severe anoxia (0% O(2)). Further study demonstrated that the degradation of mitochondrial membrane potential (MMP) was greatly inhibited and the expression of B-cell lymphoma protein-2 (Bcl-2) was increased considerably after severe anoxia in the HP groups. These results indicate that the increased anoxic tolerance, which is induced by HP in cultured hippocampal cells, may be correlated with Bcl-2 overexpression and enhanced stability of MMP, which ultimately reduces apoptosis 24 h after reoxygenation.

Involvement of increased stability of mitochondrial membrane potential and overexpression of Bcl-2 in enhanced anoxic tolerance induced by hypoxic preconditioning in cultured hypothalamic neurons.

The effects of hypoxic preconditioning (HP) on changes in mitochondrial membrane potential (MMP) and Bcl-2 expression in cultured hypothalamic neurons after severe anoxia were investigated. In the HP group, hypothalamic neurons, after a 4-day culture, were preconditioned daily under a hypoxic condition (1% O(2), 10 min) for 8 days; subsequently, the HP neurons and those in the control group (similarly cultured, but without HP) were exposed to 6 h of severe anoxia (0% O(2)). The preconditioned neurons had a higher survival rate and a lower lactate dehydrogenase leakage, compared with the control group. Although HP did not prevent the degradation of MMP during severe hypoxia, preconditioned neurons exhibited a higher level of MMP than that of the control group. Increased expression of Bcl-2 was also observed in the preconditioned hypothalamic neurons. These results suggest that HP enhances the hypoxic tolerance of hypothalamic neurons, and the underlying mechanisms may be related to the increased stability of MMP and the overexpression of Bcl-2 induced by HP.

[Protective effect of Quinacrine on striatum neurons from heat treatment injury].

AIM: To study the protective effect of Quinacrine(QA) on rat striatum neurons from the injury caused by heat environment treatment, to probe the relationship between cell membrane injury and cellular injury protection, and to seek the possibility of QA as a preventive agent to heat injury. METHODS: Primary cultured striatum neurons from newborn rats were pretreated with QA at different concentration for 1 h, and then heat-treated at 43 degrees C for another 1 h. Cell necrosis was detected by Trypan blue staining, and apoptosis was evaluated through Activated Caspase-3 dye and TdT dye. RESULTS: Heat treatment effected the survival of striatum neurons and resulted in great number of cell death, which was mainly mediated by cell necrosis process. It was shown that treatment of QA itself had little effect on the survival of striatum neurons, while QA pretreatment decreased cellular necrosis caused by following heat treatment. CONCLUSION: QA protects striatum neurons from heat environment injury at about 20 pmol/L, and the protection may mediated by reduction of necrosis.

[Effects of hypoxia on the proliferation of embryonic stem cells].

AIM: To investigate the effects of hypoxia on the proliferation of mouse embryonic stem cells (mouse ES cells) in vitro. METHODS: We observed the proliferation of ES cells by hematometery and BrdU-labeled flow cytometry (FCM), and we also detected the expression of hypoxia inducible factor-1a (HIF-1a) by RT-PCR. RESULTS: (1) The number of ES cells after culturing in the hypoxia environment (3% O2 and 10% O2) for 24 hours were lesser than those in normoxia (20% O2). (2) The number of ES cells significantly increased after intermittent hypoxia (3% O2) stimulus for 10 minutes per day for 4 days. (3) We also observed the relation between the expression of HIF-1a and the proliferation of ES cells by RT-PCR. The results showed that the expression of HIF-1a had no significant change after ES cells were culturing in hypoxia environment (3% O2 and 10% O2) for 24 hours or in intermittent hypoxia (3% O2 and 10% O2) for 4 days. CONCLUSION: These results suggest that intermittent hypoxia (3% O2) can significantly promote the proliferation of ES cells in vitro, while persistent hypoxia inhibits those, and the mechanism of these should be addressed in further.

[Glutamate-neuroexcitotoxicity protective study of monoclonal antibody against human NMDA receptor key subunit].

OBJECTIVE: To study if monoclonal antibody against human NMDA receptor key subunit (NR1) may protect neurons from excitotoxicity. METHODS: We cultured primary hippocampal neurons from 10 newborn rats and made the glutamate excitotoxicity model, examined the ratio of surviving neuron (Trypan blue dye staining) and LDH assay to study the protective effects of mAbN1, There were 2 plates in parallel per group, the experiments were repeated 4 times. RESULTS: 0.3 micro mol/L mAbN1 could protect cultured hippocampal neurons from neuroexcitotoxicity induced by 500 micro mol/L glutamate with increased survival rate 35%. Epitope peptide could block the protection of mAbN1, which suggests that the protection of mAbN1 is specific. CONCLUSION: mAbN1 is a novel NMDAR blocker with in vitro neuroprotective activity which may provide a basis for antibody therapy of neuroexcitotoxicity.

[Effects of naloxone on rat cortical neuron apoptosis induced by anoxia].

OBJECTIVE: To investigate the damage of anoxia on the cultured rat's cortical neurons and the protective effects of naloxone. METHODS: Cortical neurons cultured for 12 days were randomly divided into three groups: control, anoxic group and anoxic group plus naloxone treated. Cortical neurons were exposed to anoxic environment for 6 hours and then cultured for 24 hours under normoxic condition. The apoptosis of neurons was detected with the method of TdT-mediated dUTP nick end labeling (TUNEL) and the apoptotic rate of neurons was measured using flow cytometry. RESULTS: Anoxia could lead to increasing apoptosis and death of rate cortical neurons. Naloxone could reduce neuron apoptosis. There was a significant difference between the anoxic group and anoxic group plus naloxone (P<0.01). CONCLUSION: Naloxone can ameliorate the toxicity damage of neurons caused by anoxia through reducing neuron apoptosis, which suggests that naloxone could protect rat's cortical neurons from damage under anoxia condition.

[Effects of oxygen-glucose deprivation on cultured rat hippocampal neurons].

AIM: To investigate the effects of oxygen-glucose deprivation on cultured rat hippocampal neurons. METHODS: The hippocampal neurons cultured for 12 d were exposed to combined oxygen-glucose deprivation for 0.5 - 4 h and then cultured with original medium in normoxia for 28 h. Necrotic neurons were identified by 0.4% trypan blue staining and apoptotic neurons were detected by a TUNEL technique. Meanwhile, the area, perimeter and circle diameter of cell bodies were measured respectively by a photography analysis system. RESULTS: The percentage of necrotic cells in cultured hippocampal neurons increased significantly during oxygen-glucose deprivation, but the percentage of apoptotic cells increased significantly after 28 h oxygen-glucose recovery. Photography analysis showed that area, perimeter and circle diameter of the necrotic cell bodies were larger than those of the apoptotic ones. CONCLUSION: Oxygen-glucose deprivation can lead to severe damage of cultured hippocampal neurons. The necrosis is major during acute oxygen-glucose deprivation, while the apoptosis is major 28 h after oxygen-glucose recovery.

[Effect of CoCl2 pretreatment on Na + and K+ currents of the rat hippocampal neurons after acute hypoxia].

AIM: To study effect of CoCl2 pretreatment on the voltage-gated Na+ and K+ currents of the rat hippocampal neurons after acute hypoxia. METHODS: Primarily cultured hippocampal neurons were divided into CoCl2 pretreated and non-pretreated groups. Patch clamp whole cell recording technique was used to examine Na+ and K+ currents of the hippocampal neurons. RESULTS: After acute hypoxia, I(Na) and I(K) of the hippocampal neurons were significantly decreased and the threshold of I(Na) was right-shifted. Pretreatment of the neurons with CoCl2 inhibited the reduction of I(Na) and I(K). CONCLUSION: CcCl2 pretreatment alleviates the acute hypoxia-induced changes of I(Na) and I(K), which may be one of the mechanisms for the protective effect of CoCl2 on neurons.

[Establishment of the model of oxygen-glucose deprivation in vitro rat hippocampal neurons].

AIM: To establish the model of oxygen-glucose deprivation in vitro rat hippocampal neurons. METHODS: The hippocampal neurons cultured for 12 d were exposed to combined oxygen-glucose deprivation for 0.5-4 h and then cultured with original medium in normoxia for 24 h. Auto-biochemical analyzer determined LDH activity. The change of neuronal morphology and neuron survival were observed by converted contrast microscope and assessed by photography analysis system. Neuron apoptosis was detected by using the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nickel end labeling (TUNEL) method. RESULTS: The neurons swelled, LDH release increased and neuron survival decreased after gradually oxygen-glucose deprivation. The percentage of apoptosis increased obviously 24 h after recovering the supply of oxygen and glucose. CONCLUSION: The model of oxygen-glucose deprivation in vitro rat hippocampal neurons is established successfully by using the modified ACSF (artificial cerebral spinal fluid) with serum and glucose free.

[CoCl2-induced enhancement of glucose transport activity in mediating hypoxic tolerance in cultured hippocampal neurons].

The effect of CoCl(2) pretreatment on glucose transport activity of cultured newborn rat hippocampal neurons and its role in neuronal hypoxic tolerance were observed. The results showed that the 2-deoxy-D-[1-(3)H ]glucose uptake rate and the mRNA expressions of glucose transporters (GLUT1 and GLUT3) in the hippocampal neurons were significantly increased after a 24-hour pretreatment with CoCl(2). The cell injury induced by 6-hour or 8-hour hypoxic exposure was also greatly reduced by CoCl(2) pretreatment. The protective effect of CoCl(2) on the neurons was largely abolished by cytochalasin B, a specific inhibitor of glucose transporters. The results suggest that CoCl(2) can increase mRNA expressions of GLUT1 and GLUT3 and glucose transporter activity of the neurons, which may be an important mechanism for the increased tolerance of the neurons to hypoxia.

[Effects of rhIL-6 on Bcl-2 and Bax expression and apoptosis after anoxia-reoxygenation in cultured rat hippocampal neurons].

The purpose of the present study was to determine the effects of recombinant human interleukin-6 (rhIL-6) on the Bcl-2 and Bax expression and apoptosis after anoxia-reoxygenation in cultured rat hippocampal neurons. The control and rhIL-6 treated hippocampal neurons cultured for 12 d were exposed to anoxia environment (90% N2+10% CO2) for 2 and 4 h and then were reoxygenated for 24 and 72 h. The expression of Bcl-2 and Bax was revealed immunocytochemically using the antiserum against Bcl-2 and Bax. The apoptosis was examined by the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nickel end labeling (TUNEL) method and flow cytometric analysis. The results showed that in cultured hippocampal neurons the Bcl-2 expression decreased while Bax expression and the percentage of apoptotic neurons increased after anoxia-reoxygenation compared with those before anoxia. In comparison with the control, after anoxia-reoxygenation the Bcl-2 expression in hippocampal neurons was higher than that in rhIL-6 group; however the Bax expression and the percentage of the apoptosis were decreased in rhIL-6 group. It is suggested that rhIL-6 may play a role in protecting neurons from the damage induced by anoxia-reoxygenation.

[Effects of hypoxic-preconditioning on anoxic-tolerance and Jun expression in cultured rat hippocampal neurons].

AIM: To study the effects of hypoxic preconditioning on anoxic tolerance and Jun expression in cultured rat hippocampal neurons after anoxia/reoxygenation. METHODS: 12 day cultured hippocampal neurons in control and hypoxic preconditioning group were exposed to anoxic environment (0.90L/L N2 + 0.10 L/L CO2) for 4 h, and then reoxygenated for either 24 h or 72 h. The neurons were immunocytochemically stained using the antiserum against Jun. The number of survival neurons and the percentage of Jun expressing neurons were investigated. RESULTS: The percentage of Jun expressing neurons induced by anoxia in hypoxic-preconditioning group was significantly less than that in control group. The number of survival neurons was more in the hypoxic-preconditioning group than that in control group after anoxic reoxygenation. CONCLUSION: Hypoxic-preconditioning can induce the development of anoxic-tolerance in cultured hippocampal neurons. The decrease in Jun expressing neurons in hippocampus may be an adaptive reaction to acute anoxia.

[Relationship between enhanced anoxic tolerance induced by hypoxic preconditioning and Na+, K+ currents in cultured hypothalamic cells].

AIM: To investigate the relationship between enhanced anoxic tolerance induced by hypoxic preconditioning and Na+, K+ currents. METHODS: After hypoxic preconditioning and acute anoxia the I(Na), I(K) were measured in cultured hypothalamic cells by patch-clamp whole cell recording technique. RESULTS: The amplification of Na+ currents did not been significantly changed, but the amplification of K+ currents was in hypoxic preconditioning neurons; acute anoxia lead to the inhibition of Na+, K+ currents in the two groups, while Na+, K+ currents in non-preconditioned control group were inhibited severity than hypoxic preconditioning group. CONCLUSION: It is presumed enhanced anoxia tolerance induced by hypoxic preconditioning may be related to the opening of K+ channels.

[Effects of anoxia/reoxygenation on Fos and Jun expression and apoptosis in cultured rat hippocampal neurons].

AIM: To investigate the effects of anoxia/reoxygenation on Fos and Jun expression and apoptosis in cultured rat hippocampal neurons. METHODS: The hippocampal neurons cultured for 12 d were exposed to anoxia environment (90% N2 + 10% CO2) for 4 h and then reoxygenated for 24 h and 72 h. The neurons were immunocytochemically stained using the antiserum against Fos and Jun, and the apoptosis were detected by using the terminal deoxynucleotidyl transferase mediated biotinylated deoxyuridine triphosphate nickel end labeling (TUNEL) method and flow cytometric analysis. RESULTS: The percentage of Fos and Jun positive neurons and apoptosis neurons in cultured hippocampal neurons after anoxia/reoxygenation increased than those in control. CONCLUSION: The occurrence of neurons apoptosis is related to the increase in Fos and Jun expression in cultured hippocampal neurons after anoxia/reoxygenation.