Effects of basic fibroblast growth factor on neurogenesis in the subventricular zone of 3-day-old rats following brain ischemia

The subventricular zone (SVZ), lining the lateral ventricular system, is the largest germinal region in mammals. In there, neural stem cells express markers related to astoglial lineage that give rise to new neurons and oligodendrocytes in vivo. In the adult human brain, in vitro evidence has also shown that astrocytic cells isolated from the SVZ can generate new neurons and oligodendrocytes. These proliferative cells are strongly controlled by a number of signals and molecules that modulate, activate or repress the cell division, renewal, proliferation and fate of neural stem cells. In this review, we summarize the cellular composition of the adult human SVZ (hSVZ) and discuss the increasing evidence showing that some trophic modulators strongly control the function of neural stem cells in the SVZ.

Objective To investigate the effects of functional electrical stimulation (FES) on endogenous proliferation of the neural stem cells within the brain and the behaviors in rats with acute cerebral infarction and explore the FES therapeutic mechanism on improving the neural function after the cerebral infarction. Methods Fifty-four SD adult male rats were randomly allocated into FES treatment group, placebo stimulation group and sham-operated group (n=18). Focal cerebral infarction models were induced by the performent of middle cerebral artery occlusion (MCAO) in rats; the FES treatment group began receiving the FES (10 min/d, once dairy) and the placebo stimulation group did not give any special treatment since the 3rd day of the successful model inducement. The expression of nestin positive cells in the hippocampus subgranular zone and subventricular zone was examined by immunohistochemistry staining and the expression of nestin protein at ischemia side was detected by Western blot analysis on the 3rd, 7th and 14th d after MCAO; meanwhile, the behavior functions of rats at various time points were evaluated. Results The number of nestin positive cells in the hippocampus subgranular zone and subventricular zone and the expression of nestin protein from ischemia side brain in the FES treatment group significantly increased than those in the placebo stimulation group on the 7th and 14th d after MCAO (P<0.05). And statistical difference was noted on the 14th d on the behavior functional evaluation between the FES treatment group and the placebo stimulation group (P<0.05). Conclusion FES may enhance the endogenous proliferation of the neural stem cells within the brain in rats with acute cerebral infarction and improve the behavior function in rats, which may be one of the mechanisms of FES on improving the neural function after cerebral infarction. Key words: Functional electrical stimulation; Neural stem cell; Cerebral infarction; Nestin

Neurogenesis in the adult rat brain after intermittent hypoxia

Objective To investigate the effects of melatonin on the proliferation of neural stem cells (NSCs) in cerebral ischemia reperfusion (IR) rats, and to explore the possible mechanisms. Methods Seventy-two rats were randomly divided into the normal control group (n=12), model group (n=30) and melatonin group (n=30) according to the random number table. The rats in the model group and melatonin group were divided into four subgroups: 6 h, 24 h, 72 h and 7 d subgroups according to the time after IR. The morphological changes of the subventricular zone (SVZ) were examined by HE staining; the effects of melatonin on NSCs proliferation were examined by immunofluorescence staining; the effects of melatonin on toll-like receptor 4 (TLR4) and nuclear factor (NF) -κB p65 protein were examined by immunohistochemistry staining and Western blotting analysis. The correlation between the proliferating NSCs and TLR4 protein or the NF-κB p65 protein was analyzed by linear regression analysis. Results HE staining showed that the cells in the SVZ of rats in the model group were in disorder and irregular in shape. In the melatonin group, the cells in the SVZ of the injured side were relatively well arranged. Immunofluorescence staining showed that the number of proliferating cell nuclear antigen (PCNA)+Nestin+4′,6-diamidino-2-phenylindole (DAPI)+ cells in the SVZ of the model (498.47±26.44/mm2) and melatonin groups (623.10±39.70/mm2) increased gradually, and reached a higher level after IR for 7 days, which were significantly higher than the normal control group (203.91±32.23/mm2) (F=35.193, 170.344, 277.536, 285.947, all P<0.01). The number of PCNA+Nestin+DAPI+ cells in the melatonin group rats at each time points was significantly higher than that in the model group (F=102.561, 91.244, 168.502, 38.013, all P<0.01). Immunohistochemistry staining showed that the numbers of TLR4+ and NF-κB p65+ cells in the SVZ of the model (740.02±31.63/mm2; 710.01±26.59/mm2) and melatonin groups (555.57±25.28/mm2; 528.85±30.60/mm2) increased gradually, and reached a higher level 7 d after IR, which were significantly higher than the normal control group (107.97±12.84/mm2; 109.80±13.89/mm2) (F=21.413, 263.059, 873.691, 1 037.098, all P<0.01; F=26.374, 372.940, 854.826, 929.018, all P<0.01). There were less TLR4+ (F=7.641, 25.135, 66.094, 103.753, all P<0.05) and NF-κB p65+ cells (F=18.612, 69.597, 113.113, 119.814, all P<0.01) in the melatonin group as compared with those in the model group at each time points. Western blotting analysis showed that the expression of TLR4 (0.87±0.08; 0.68±0.06) and NF-κB p65 (0.72±0.05; 0.58±0.05) protein was higher in the model and melatonin groups as compared with the normal control group (0.35±0.04, 0.31±0.03; F=107.43, F=132.51, both P<0.01). The expression of the TLR4 and NF-κB p65 protein was lower in the melatonin group as compared with that in the model group (P<0.01). Linear regression analysis showed that the differences of PCNA+Nestin+DAPI+ cells were all negatively correlated with that of the TLR4+ cells and NF-κB p65+ cells in the melatonin group (r2=0.838, r2=0.813, both P<0.01). Conclusion Melatonin can inhibit the expression of TLR4 and NF-κB p65 protein, thus promote the proliferation of endogenous NSCs in cerebral ischemia reperfusion rats. Key words: Brain ischemia; Reperfusion injury; NF-kappa B; Neural stem cells

miR-17∼92 exerts stage-specific effects in adult V-SVZ neural stem cell lineages.

To compare the repair effects of different doses of human umbilical cord mesenchymal stem cells (hUC-MSCs) on white matter injury (WMI) in neonatal rats. Two-day-old Sprague-Dawley neonatal rats were randomly divided into five groups: sham operation group, WMI group, and hUC-MSCs groups (low dose, medium dose, and high dose), with 24 rats in each group. Twenty-four hours after successful establishment of the neonatal rat white matter injury model, the WMI group was injected with sterile PBS via the lateral ventricle, while the hUC-MSCs groups received injections of hUC-MSCs at different doses. At 14 and 21 days post-modeling, hematoxylin and eosin staining was used to observe pathological changes in the tissues around the lateral ventricles. Real-time quantitative polymerase chain reaction was used to detect the quantitative expression of myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) mRNA in the brain tissue. Immunohistochemistry was employed to observe the expression levels of GFAP and neuron-specific nuclear protein (NeuN) in the tissues around the lateral ventricles. TUNEL staining was used to observe cell apoptosis in the tissues around the lateral ventricles. At 21 days post-modeling, the Morris water maze test was used to observe the spatial learning and memory capabilities of the neonatal rats. At 14 and 21 days post-modeling, numerous cells with nuclear shrinkage and rupture, as well as disordered arrangement of nerve fibers, were observed in the tissues around the lateral ventricles of the WMI group and the low dose group. Compared with the WMI group, the medium and high dose groups showed alleviated pathological changes; the arrangement of nerve fibers in the medium dose group was relatively more orderly compared with the high dose group. Compared with the WMI group, there was no significant difference in the expression levels of MBP and GFAP mRNA in the low dose group (P>0.05), while the expression levels of MBP mRNA increased and GFAP mRNA decreased in the medium and high dose groups. The expression level of MBP mRNA in the medium dose group was higher than that in the high dose group, and the expression level of GFAP mRNA in the medium dose group was lower than that in the high dose group (P<0.05). Compared with the WMI group, there was no significant difference in the protein expression of GFAP and NeuN in the low dose group (P>0.05), while the expression of NeuN protein increased and GFAP protein decreased in the medium and high dose groups. The expression of NeuN protein in the medium dose group was higher than that in the high dose group, and the expression of GFAP protein in the medium dose group was lower than that in the high dose group (P<0.05). Compared with the WMI group, there was no significant difference in the number of apoptotic cells in the low dose group (P>0.05), while the number of apoptotic cells in the medium and high dose groups was less than that in the WMI group, and the number of apoptotic cells in the medium dose group was less than that in the high dose group (P<0.05). Compared with the WMI group, there was no significant difference in the escape latency time in the low dose group (P>0.05); starting from the third day of the latency period, the escape latency time in the medium dose group was less than that in the WMI group (P<0.05). The medium and high dose groups crossed the platform more times than the WMI group (P<0.05). Low dose hUC-MSCs may yield unsatisfactory repair effects on WMI in neonatal rats, while medium and high doses of hUC-MSCs have significant repair effects, with the medium dose demonstrating superior efficacy.

Ischemic stroke stimulates neurogenesis in the adult rodent brain. The molecules underlying stroke-induced neurogenesis have not been fully investigated. Using real-time reverse transcription-PCR, we found that stroke substantially up-regulated angiopoietin 2 (ANG2), a proangiogenic gene, expression in subventricular zone neural progenitor cells. Incubation of neural progenitor cells with recombinant human ANG2 significantly increased the number of beta-III tubulin-positive cells, a marker of immature neurons, but did not alter the number of glial fibrillary acidic protein (GFAP)-positive cells, a marker of astrocytes, suggesting that ANG2 promotes neuronal differentiation. Blockage of the ANG2 receptor, Tie2, with small interference RNA (siRNA)-Tie2 attenuated recombinant human ANG2 (rhANG2)-increased beta-III tubulin mRNA levels compared with levels in the progenitor cells transfected with control siRNA. Chromatin immunoprecipitation analysis revealed that CCAAT/enhancer-binding protein (C/EBP beta) up-regulated by rhANG2 bound to beta-III tubulin, which is consistent with published data that there are several C/EBP beta binding sites in the promoter of beta-III tubulin gene. In addition, rhANG2 enhanced migration of neural progenitor cells measured by single neurosphere assay. Blockage of Tie2 with siRNA-Tie2 and a Tie2-neutralizing antibody did not suppress ANG2-enhanced migration. However, inhibition of matrix metalloproteinases with GM6001 blocked ANG2-enhanced migration. Collectively, our data suggest that interaction of ANG2, a proangiogenic factor, with its receptor Tie2 promotes neural progenitor cell differentiation into neuronal lineage cells, whereas ANG2 regulates neural progenitor cell migration through matrix metalloproteinases, which do not require its receptor Tie2.

To study growth characteristics of neural stem cells (NSCs) from subventricular zone (SVZ) of the different human fetal brain at different gestational age and to provide experimental and theoretical evidences for clinical application of NSCs for treatment of certain diseases. Ninety human embryos at gestational age 16 - 36 weeks were collected and were divided into six groups according to gestational age: 16 w, 20 w, 24 w, 28 w, 32 w and 36 w. Each group had 15 embryos and brain tissues were taken from each embryo's SVZ. All subjects had congenital heart disease or digestive tract abnormity diagnosed with B ultrasound at antepartum, but none had abnormal development of brain. Pregnant mother and her husband desire termination of pregnancy. The morphology, existing mode and the number of neural stem cells in subventricular zone were examined with immunohistochemical method. The NSCs in subventricular zone were cultured, passaged and differentiated with cell culture technique, then were identified with immunohistochemical method. NSCs in SVZ from the different human fetal brain existed in a scattered manner in the network formed by stellate cells, NSCs had round, ellipse and fusiform shape, especially in stellate shape. NSCs had larger and smaller size and distributed in dense or scattered forms, each having zero to two enations, most had one or two. NSCs had less cytoplasm. The nucli of the NSCs had a round shape with loose chromatin and 1 - 4 nucleoli. Most of NSCs existed in singular scattered form, some of them showed symmetrical or asymmetrical division, some of them showed synaptic connection with other NSCs. The number of NSCs in SVZ from groups with different fetal age decreased with increasing gestational age (chi(2) = 4644.602, P < 0.01). NSCs in SVZ from the different human fetal brain cultured with serum-free medium formed typical neurospheres in suspension. The cells could be passaged continuously, and could express nestin antigen. Serum-contained medium induced neural stem cells to differentiate and express specific antigens of neuron, astrocyte and oligodendrocyte. NSCs existed in SVZ of human embryos at different gestational age. There are differences in morphology, existing pattern and the number of NSCs in SVZ at different gestational age. NSCs in SVZ at different gestational age may be cultured in vitro.

Objective To detect the effect of olfactory bulb(OB)lesion on proliferation,migration and differentiation of the neural stem cells(NSCs)in the subventricular zone(SVZ).Methods Forty-two healthy female SD rats were enrolled and randomly divided into normal control group,isotonic saline group and OB lesion at day 3,at weeks 1,2,3 and 4 groups,six rats per group.OB lesion was induced by N-methyl-D-aspartic acid(NMDA)injection.Bromodeoxyuridine(BrdU)was injected intraperitoneally to label NSCs.Immunohistochemical staining was used to detect the proliferation of SVZ NSCs.In addition,another 18 rats were randomly divided into normal control group,isotonic saline group and lesion group,six rats per group.BrdU was injected intraperitoneally one week after OB lesion and then the animals were sacrificed four weeks after BrdU injection to detect the migration and differentiation of NSCs with immunohistochemistry and immunofluorescence.Results Three days after OB lesion,BrdU-positive cells in SVZ began to increase(26.33 ± 2.58,P ＜0.01),reached the maximum at week 1 (35.33 +3.01,P＜0.01)and still sustained a high level at week 4(19.50+ 2.26,P＞0.05).Five weeks after the OB lesion,the rostral migratory-stream(RMS)and the BrdU-positive cells in OB were significantly increased(86.50 + 5.09,P ＜ 0.01)and(52.83 + 3.87,P ＜ 0.01),respectively.By using fluorescence double staining,most of the BrdU-positive cells were co-localized with the neuronal nuclear antigen(Neun),with a portion of BrdU-positive cells expressing the glial fibrillary acidic protein (GFAP).Conclusions OB lesion can improve the proliferation of NSCs in SVZ and migration of NSCs to OB.The newborn cells can differentiate into not only neurons,but also gliocytes and may be involved in lesion repair. Key words: Olfactory bulb; Neural stem cell; Cell proliferation; Rats

The accumulation of the intermediate filament protein, glial fibrillary acidic protein (GFAP), in astrocytes of Alexander disease (AxD) impairs proteasome function in astrocytes. We have explored the molecular mechanism that underlies the proteasome inhibition. We find that both assembled and unassembled wild type (wt) and R239C mutant GFAP protein interacts with the 20 S proteasome complex and that the R239C AxD mutation does not interfere with this interaction. However, the R239C GFAP accumulates to higher levels and forms more protein aggregates than wt protein. These aggregates bind components of the ubiquitin-proteasome system and, thus, may deplete the cytosolic stores of these proteins. We also find that the R239C GFAP has a greater inhibitory effect on proteasome system than wt GFAP. Using a ubiquitin-independent degradation assay in vitro, we observed that the proteasome cannot efficiently degrade unassembled R239C GFAP, and the interaction of R239C GFAP with proteasomes actually inhibits proteasomal protease activity. The small heat shock protein, alphaB-crystallin, which accumulates massively in AxD astrocytes, reverses the inhibitory effects of R239C GFAP on proteasome activity and promotes degradation of the mutant GFAP, apparently by shifting the size of the mutant protein from larger oligomers to smaller oligomers and monomers. These observations suggest that oligomeric forms of GFAP are particularly effective at inhibiting proteasome activity.

Progenitors in the telencephalic subventricular zone (SVZ) remain mitotically active throughout life, and produce different cell types at embryonic, postnatal and adult stages. Here we show that Mash1, an important proneural gene in the embryonic telencephalon, is broadly expressed in the postnatal SVZ, in progenitors for both neuronal and oligodendrocyte lineages. Moreover, Mash1 is required at birth for the generation of a large fraction of neuronal and oligodendrocyte precursors from the olfactory bulb. Clonal analysis in culture and transplantation experiments in postnatal brain demonstrate that this phenotype reflects a cell-autonomous function of Mash1 in specification of these two lineages. The conservation of Mash1 function in the postnatal SVZ suggests that the same transcription mechanisms operate throughout life to specify cell fates in this structure, and that the profound changes in the cell types produced reflect changes in the signalling environment of the SVZ.

Objective To investigate the effects of functional electrical stimulation (FES) on motor function and the expression of bromodeoxyuridine (Brdu) + and glial fibrillary acid protein (GFAP) + in the subventricular zone (SVZ) of rats with acute cerebral infarction,and to explore it's mechanism. Methods A rat model of cerebral infarction was established using Longa's technique for middle cerebral artery occlusion (MCAO) with an intraluminal filament.The rats were randomly divided into a FES group,a placebo stimulation group and a control group.In each group,rats were randomly allocated into 1 d,3 d,7 d and 14 d subgroups (6 rats/subgroup).Superficial electrodes were pasted on the paralyzed forelimbs of rats in the FES group for connecting with the FES instrument,and FES treatment was carried out with a current of 4-5 mA for 15 min on the third day after the MCAO operation to produce extension of the wrist and the digits of the paralyzed forelimb.The rats in the placebo stimulation group were pasted with electrodes,but no FES was administered and they received no other treatment.Neurological deficits were evaluated using the modified neurological severity score (mNSS) before treatment and on the 1 st,3rd,7th,and 14th day after treatment. BrdU and GFAP positive cells in the SVZ were detected by immunofluorescence techniques.Results After 7 or 14 days the motor function of rats in the FES group had improved significantly compared with the placebo stimulation and control groups.Compared with the other two groups,the expression levels of BrdU+ and GFAP+ cells in the ischemic SVZ in the FES group were significantly higher at the 3rd,7th and 14th day.Conclusion FES can improve motor function after acute cerebral infarction and also promote the proliferation and differentiation of neural stem cells in the SVZ. Key words: Functional electrical stimulation ; Cerebral infarction ; Neural plasticity ; Subventricularzone

Basic fibroblast growth factor stimulates the proliferation and differentiation of neural stem cells in neonatal rats after ischemic brain injury

Neurogenesis in the subventricular zone (SVZ) is regulated by diffusible factors and cell–cell contacts. In vivo, SVZ stem cells are associated with the abluminal surface of blood vessels and such interactions are thought to regulate their neurogenic capacity. SVZ neural stem cells (NSCs) have been described to contact endothelial-derived laminin via α6β1 integrin. To elucidate whether heterocellular contacts with brain endothelial cells (BEC) regulate SVZ cells neurogenic capacities, cocultures of SVZ neurospheres and primary BEC, both obtained from C57BL/6 mice, were performed. The involvement of laminin-integrin interactions in SVZ homeostasis was tested in three ways. Firstly, SVZ cells were analyzed following incubation of BEC with the protein synthesis inhibitor cycloheximide (CHX) prior to coculture, a treatment expected to decrease membrane proteins. Secondly, SVZ cells were cocultured with BEC in the presence of an anti-α6 integrin neutralizing antibody. Thirdly, BEC were cultured with β1−/− SVZ cells. We showed that contact with BEC supports, at least in part, proliferation and stemness of SVZ cells, as evaluated by the number of BrdU positive (+) and Sox2+ cells in contact with BEC. These effects are dependent on BEC-derived laminin binding to α6β1 integrin and are decreased in cocultures incubated with anti-α6 integrin neutralizing antibody and in cocultures with SVZ β1−/− cells. Moreover, BEC-derived laminin sustains stemness in SVZ cell cultures via activation of the Notch and mTOR signaling pathways. Our results show that BEC/SVZ interactions involving α6β1 integrin binding to laminin, contribute to SVZ cell proliferation and stemness.

Objective To investigate the effects of functional electrical stimulation (FES) on motor function and on the expression, proliferation, migration and differentiation of endogenous neural stem cells in the subventricular zone (SVZ) after cerebral ischemia. Methods Middle cerebral artery occlusion (MCAO) was used to induce a model of cerebral ischemia in 108 rats using the modified Zea-Longa method of intraluminal filament occlusion. They were then randomly divided into an FES group, a placebo stimulation group and a control group with 36 cases in each. Superficial FES electrodes were pasted on the paralyzed forelimbs of the rats in the first two groups, though FES treatment was administered only to the FES group beginning on the 3rd day after the MCAO operation. The stimulation was designed to produce extension of the wrist and digits of the paralyzed forelimb. Before, and after 1, 3, 7 and 14 days of the treatment, the neurological deficit was evaluated using modified neurological severity scoring (mNSS). BrdU+ /GFAP+ , BrdU+ /DCX+ and BrdU+ /NeuN+ cells in the SVZ were detected using immunofluorescence technique. Results After 7 and 14 days of treatment, the average motor function of the rats in the FES group had improved significantly when compared with the averages of the other two groups. Compared with the other two groups, the average number of BrdU+ /GFAP+ positive cells in the ischemic SVZ was also significantly greater in the FES group after 7 and 14 days of treatment. After 14 days, BrdU+ /Dcx+ positive cells in the FES group had also increased significantly more, but only a few BrdU+ /NeuN+ cells had appeared in any of the three groups. Conclusion FES can improve motor function after cerebral ischemia, and promote proliferation and differentiation of neural stem cells in the SVZ. Key words: Electrical stimulation; Brain ischemia; Functional electrical stimulation; Neural stem cells; Subventricular zone