Endogenic Neurogenesis Of Cortex And Hippocampus In The Adult Rat After Traumatic Brain Injury

PartⅠ Endogenic Neurogenesis of Cortex in the Adult Rat after Traumatic BrainInjuryObjectiveTo investigate whether endogenic neurogenesis happened in the cortex in the adult ratafter traumatic brain injury (TBI) and the factors that influenced the process.MethodsExperiment in vivo:1. SD rats were randomly divided into TBI and sham groups. The rats in sham groupwere performed craniotomy only without any injury. In TBI group, rats model of TBI wasestablished by a fluid percussion device.2. Rats were injected with BrdU at1to7days post-injury (dpi) to allow identificationof newborn cells.3. Rats were sacrificed at1,3,7,14and28dpi for immunofluorescence. The neuralprogenitor cells (NPCs) were detected by nestin/sox-2. Radial glial (RG)-like cells weredetected by GFAP/sox-2. Newborn neuronal progenitor cells were detected by DCX/BrdU.Newborn neurons were detected by MAP-2/BrdU. Newborn mature neurons were detectedby NeuN/BrdU. Newborn astrocytes were detected by GFAP/BrdU. Newbornoligodendrocytes were detected by CNP/BrdU.4. The apoptosis of newborn cells after1,3,7,14and28dpi were detected byTUNEL/BrdU.5. The level of SDF-1in the injury cortex after1,3,7,14and28dpi was detected byenzyme linked immunosorbent assay (ELISA) kit.6. The level of glutamate in the cerebrospinal fluid after1,3,7,14and28dpi wasdetected by high performance liquid chromatography (HPLC).Experiment in vitro:1. The neural stem cells (NSCs) identification of neurospheres from the injury cortex after TBI was performed by immunofluorescence. Double-labeled staining of BrdU/Nestinwas used to detect their embryonic characteristics and self-proliferating ability. Staining ofGFAP, MAP-2, CNP were used to detect their capacities of multipotent differentiation.2. NR1, the N-methyl-D-aspartate (NMDA) receptors subunit was labeled byimmunofluorescence. The percentage of NR1positive NSCs was analyzed by flowcytometry.3. The above mentioned NSCs were cultured under four different conditions: NSCs ofcontrol group were cultured in the serum free DMEM/F12medium. NSCs of glutamate(Glu) group were cultured in the serum free DMEM/F12medium with glutamate (5μM).NSCs of Glu+MK-801(0h) group were cultured in the serum free DMEM/F12mediumwith glutamate (5μM) and MK-801(10μM). NSCs of Glu+MK-801(24h) were culturedin the serum free DMEM/F12medium with glutamate (5μM), after24h, MK-801(10μM)was added in the medium. After culture1,3,7,14days in vitro (DIV), the neuronaldifferentiation of NSCs was observed by DCX or MAP-2immunofluorescence and theapoptosis of cells were detected by TUNEL kit.4. After culture1,3,7,14DIV, the level of NR1mRNA of differentiated cells incontrol and Glu+MK-801(24h) groups was detected by real-time PCR. The level of NR1protein of differentiated cells of control and Glu+MK-801(24h) groups was detected byWestern Blot.5. The migration of differentiated cells was detected by Transwell assay. The abovementioned NSCs were cultured in the upper chamber and divided into control, TBI cortexextract and SDF-1groups. The serum free DMEM/F12medium was added in the bottomchamber of control group. The serum free DMEM/F12medium with TBI cortex extract(20μl/ml) was added in the bottom chamber of TBI cortex extract group. The serum freeDMEM/F12medium with SDF-1(200ng/ml) was added in the bottom chamber of SDF-1group. After3DIV，the migrated cells were observed by Hoechst staining. Whether themigrated cells was CXCR4positive was confirmed by immunofluorescence.ResultsExperiment in vivo:1. The results of brain slice immunofluorescence showed nestin+/sox-2+NPCs andGFAP+/sox-2+RG-like cells emerged in peri-injured cortex at1,3,7,14dpi and peaked at3dpi. The number of GFAP+/sox-2+cells was less than that of nestin+/sox-2+cells. Nestin+/sox-2+cells from posterior periventricle (PPV) immigrated into peri-injured cortexthrough corpus callosum (CC) were found. DCX+/BrdU+newborn immature neurons inperi-injured cortex were found only at3,7,14dpi. A few MAP-2+/BrdU+newborn neuronsin peri-injured cortex were found only at7and14dpi. NeuN+/BrdU+mature neurons werenot found in peri-injured cortex at1,3,7,14and28dpi. While GFAP+/BrdU+astrocytesemerged in peri-injured cortex at1,3,7,14,28dpi and peaked at7dpi then kept in astable state. In the corresponding time point, the percentage of GFAP+/BrdU+astrocytes inBrdU+cells was more than that of NPCs or newborn neurons. No CNP+/BrdU+oligodendrocytes were found in peri-injured cortex.2. The results of TUNEL/BrdU staining showed TUNEL+/BrdU+cells emerged inperi-injured cortex at1,3,7,14dpi and peaked at7dpi.3. The level of SDF-1in the injury cortex peaked at3dpi then decreased and wassimilar to that of sham group at14,28dpi.4. The level of glutamate in the cerebrospinal fluid peaked at1dpi then decreased andwas similar to that of sham group at14,28dpi.Experiment in vitro:1. The neurospheres from the injury cortex after TBI were BrdU+/Nestin+and coulddifferentiate into GFAP+astrocytes, MAP-2+neurons and CNP+oligodendrocytes.2. The results of flow cytometry showed43±1.06%of NSCs from injury cortex wasNR1positive.3. After culture1DIV, the number of DCX+neuronal progenitor cells in the Glu andGlu+MK-801(24h) group was more than that of control and Glu+MK-801(0h) group. Afterculture3DIV, many DCX+neuronal progenitor cells were still found in theGlu+MK-801(24h) group and the number of DCX+neuronal progenitor cells in the Glugroup decreased but was still more than that control and MK-801(0h) group. After culture7DIV many DCX+neuronal progenitor cells were still found in the Glu+MK-801(24h)group but only a few DCX+neuronal progenitor cells were found in the other three groups.After culture14DIV, few DCX+neuronal progenitor cells was found in the four groups butmany MAP-2+cells were found in the Glu+MK-801(24h) group and a few MAP-2+cellswere found in the other three groups.4. After culture1DIV, a few TUNEL+cells were found in the control, Glu,Glu+MK-801(0h) and Glu+MK-801(24h) groups. After culture3,7,14DIV, the percentage of TUNEL+cells in the control, Glu+MK-801(0h) and Glu+MK-801(24h)groups kept in a stable state but the percentage of TUNEL+cells in the Glu group peaked at3DIV then dcreased and was still more than that in the other three groups at thecorresponding time.5. The expression of NR1mRNA in the Glu+MK-801group increased gradually andpeaked at7DIV then kept in a sable state. The expression of NR1mRNA in the controlgroup almost kept in a sable state. The level of NR1mRNA in the Glu+MK-801group wasmore than that of control group at the corresponding time.6. The expression of NR1protein in the Glu+MK-801group increased gradually andpeaked at7DIV then kept in a sable state. The expression of NR1protein in the controlgroup almost kept in a sable state. The level of NR1protein in the Glu+MK-801group wasmore than that of control group at the corresponding time.7. The number of migrated cells in the Extract and SDF-1groups was more than thatof control group. Most of the migrated cells were CXCR4positive.Conclusion：NPCs emerged in peri-injured cortex of adult rats after TBI. Some of them originatedin the local reactive RG–like cells, and some of them came from PPv, which maybe wasrelative to the high expression of SDF-1in the injury cortex after TBI. The NPCs coulddifferentiate into immature neurons and astrocytes, but the former failed to grow up tomature neurons. The enhancement of glutamate after TBI may affect the destination ofimmature neurons which maybe result from increasing expression of NMDA in theneuronal development. PartⅡ Effect of Oxiracetam on Neurogenesis in the Hippocampus after TraumaticBrain InjuryObjective：To investigate the effect of oxiracetam on neurogenesis in the hippocampus after TBIand explore its mechanism.Methods：Experiments in vivo: 1. The SD rats were trained by Morris Water Maze test then were divided into controlgroup, NS group and oxiracetam group. The NS group and oxiracetam group, rat model ofTBI was established by a fluid percussion device. The rat model of NS group injected with1ml NS at1to14dpi through caudal vein. The rats model in oxiracetam groups wereinjected with1ml oxiracetam (200mg/kg) at1to14dpi through caudal vein. The rats incontrol group were not given any treatment.2. Place navigation test was performed at15to18dpi and spce probe trial test wasperformed at19dpi.3. The newborn neuronal progenitor cells in the dentate gyrus were observed by DCXimmunofluorescence at20dpi. The cholinergic neurons in the septal area and Meynertbasal nuclei were detected by ChAT immunofluorescence at20dpi.4. The level of ACh in the hippocampus was detected by ELISA kit at20dpi.5. The level of ATP in the hippocampus was detected by ATP kit at20dpi.Experiments in vitro：1. Hippocampal NSCs were prepared from pregnant SD rat embryos on E14-15. Aftertwo passages, NSCs were cultured under three different conditions: NSCs of control groupwere cultured in the serum free DMEM/F12medium. NSCs of NS group were cultured inthe serum free DMEM/F12medium with NS (10μl/ml). NSCs of oxiracetam group werecultured in the serum free DMEM/F12medium with oxiracetam (4mg/ml). After3DIV,the differentiated neuronal progenitor cells were detected by DCX immunofluorescence.The ATP level of cells was detected by ATP kit.2. Single cell from basal forebrain of pregnant SD rat embryos on E14-15wascultured in the bottom chamber of Tanswell24-well plate under three different conditions:the serum free DMEM/F12medium was added in the cholinergic neuron group; the serumfree DMEM/F12medium with NS (10μl/ml) was added in the NS+cholinergic neurongroup; the serum free DMEM/F12medium with oxiracetam (4mg/ml) was added in theoxiracetam+cholinergic neuron group. Only serum free DMEM/F12medium was added inthe bottom chamber of well as a control group. Then the hippocampal NSCs were culturedin the upper chamber of four groups. After co-culture3DIV, the differentiated cholinergicneurons in the bottom chamber were observed by ChAT immunofluorescenc and thedifferentiated neuronal progenitor cells were detected by DCX immunofluorescence. Thelevel of ACh in the culture medium was detected by ELISA kit. 3. Single cell from basal forebrain of pregnant SD rat embryos on E14-15wascultured in24-well plates and divided into control, NS+TBI cortex extract andoxiracetam+TBI cortex extract groups: the cells in control group were still cultured inthe serum free DMEM/F12medium; the cells in NS+TBI cortex extract group werecultured in the serum free DMEM/F12medium with NS (10μl/ml) and TBI cortex extract(20μl/ml); the cells of oxiracetam+TBI cortex extract group were cultured in the serumfree DMEM/F12medium with oxiracetam (4mg/ml) and TBI cortex extract (20μl/ml).After3DIV, the apoptosis of cells was detected by TUNEL kit.Results：Experiments in vivo:1. The escape latency of rats in the oxiracetam and control groups was less than thatof NS group.2. The number of platform pass of rats in the oxiracetam and control groups was morethan that of NS group.3. A few DCX+neuronal progenitor cells were found in the dentate gurus of controlgroup. Many DCX+neuronal progenitor cells were found in the dentate gurus ofoxiracetam and NS groups but the number of DCX+neuronal progenitor cells ofoxiracetam group was than that of NS group.4. Many cholinergic neurons were found in the septal area and Meynert basal nucleiof control group. The number of cholinergic neurons in the oxiracetam group decreasedslightly and was more than that of NS group.5. The level of hippocampus ACh in oxiracetam group was less than that of controlgroup but more than that of NS group.6. The level of hippocampus ATP in oxiracetam group was less than that of controlgroup but more than that of NS group.Experiments in vitro:1. A few DCX+neuronal progenitor cells were found in the control, NS andoxiracetam groups. The difference of DCX+neuronal progenitor cells number in the3groups was not statistically significant.2. The ATP level in cells of oxiracetam group was more than that of control and NSgroups.3. Many ChAT+cholinergic neurons were found in cholinergic neuron, NS+ cholinergic neuron, oxiracetam+cholinergic neuron groups. The number difference ofChAT+cholinergic neurons in the three groups was not statistically significant. But theChAT fluorescence intensity of cholinergic neurons in the oxiracetam+cholinergic neurongroup was higher than that of cholinergic neuron and NS+cholinergic neuron group. Nopositive cells were found in the control group.4. DCX+neuronal progenitor cells were found in the control, cholinergic neuron, NS+cholinergic neuron, oxiracetam+cholinergic neuron groups. The number of DCX+neuronalprogenitor cells in the oxiracetam+cholinergic neuron group was more than that of theother three groups. The number of DCX+neuronal progenitor cells in the cholinergicneuron and NS+cholinergic neuron groups than that of control group.5. The ACh level in the culture medium of oxiracetam+cholinergic neuron group wasmore than that of cholinergic neuron and NS+cholinergic neuron groups. ACh was notdetected in the culture medium of control group.6. Many TUNEL+cells were found in the NS+TBI cortex extract group while lessTUNEL+cells were found in the control and oxiracetam+TBI cortex extract groups.Conclusion：Oxiracetam could promote the endogenic neurogenesis of hippocampus in the adultrat after TBI and improve the cognition recovery. Oxiracetam could protect the basalforebrain cholinergic system after TBI and promote ACh secretion to enhance theneurogenesis of hippocampus. Oxiracetam also could enhance the level of ATP inhippocampal cells to improve the neurogenesis of hippocampus.