Objectives: To study the effects of Porphyromonas gingivalis (Pg) injected through tail vein on the molecular expression levels of biomarkers of neural stem cells (NSC) and neurons in the hippocampus of wild-type adult rats, and the effects on hippocampal neurogenesis. Methods: Eighteen male Sprague-Dawley (SD) rats were randomly divided into 3 groups based on the table of random numbers (n=6 in each group). In low-intensity group and high-intensity group, rats were injected intravenously through tail vein with 200 μl Pg ATCC33277 [1.0×10³ and 1.0×10⁸ colony forming unit (CFU), respectively] 3 times per week for 8 weeks. In the sham group, 200 μl of phosphate buffer saline (PBS) was given instead. Behavioral tests: the navigation and the exploration tests using Morris water maze (MWM) were applied to evaluate learning and memory ability of rats. Immunohistochemistry was performed to detect cells positively expressing nestin, doublecortin (DCX) and neuronal nuclei (NeuN) in the subgranular zone (SGZ) of rats in each group. Western blotting was used to evaluate the expression levels of nestin, DCX and NeuN in rat hippocampus. Results: Learning and memory abilities: on day 5 of navigation test, the lagency time was 22.83 (16.00, 38.34) s in the high-intensity group, significantly longer than the sham group [5.59 (5.41, 6.17) s] (t=-11.17, P<0.001). There were no significant differences between the low-intensity group [9.85 (8.75, 21.01) s] and the sham group (t=-6.83, P=0.080). Results in the exploration test showed that, in the high-intensity group, the number of fime crossing over the previous platform area within 60 s was 1.50 (1.00, 2.00), significantly less than the sham group [4.00 (2.75, 4.00)] (t=9.75, P=0.003); no significant differences between the low-intensity group [2.50 (2.00, 3.00)] and the sham one (t=4.50, P=0.382). Immunohistochemistry showed that the nestin⁺ cell density in the low-intensity group [(35.36±4.32) cell/mm²] and high-intensity group [(26.51±5.89) cell/mm²] were significantly lower than the sham group [(59.58±14.15) cell/mm²] (t=24.21, P=0.018; t=33.07, P=0.005); as for the mean absorbance of DCX⁺ cells, the low-intensity group (0.007±0.002) and the high-intensity group (0.006±0.002) were significantly lower than the sham group (0.011±0.001) (t=0.004, P=0.018; t=0.006, P=0.005); compared with the sham group [(1.13±0.14)×10³ cell/mm²], the density of NeuN⁺ neurons in the high-intensity group [(0.75±0.08)×10³ cell/mm²] was significantly reduced (t=0.38, P=0.017), and was not significantly changed in the low-intensity group [(0.88±0.19)×10³ cell/mm²] (t=0.25, P=0.075). Western blotting results showed that, compared with the sham group, the expression levels of nestin, DCX, and NeuN were significantly reduced in the high-intensity group (t=0.74, P<0.001; t=0.18, P=0.014; t=0.35, P=0.008), but were not statistically changed in the low-intensity group (t=0.18, P=0.108; t=0.08, P=0.172; t=0.19, P=0.077). Conclusions: Pg injected through tail vein may reduce learning and memory abilities of wild-type rats, and may reduce the number of nestin, DCX, and NeuN-positive cells, and the protein expression levels of the above molecules in the hippocampus.
Animals ; Biomarkers/metabolism* ; Hippocampus/metabolism* ; Male ; Nestin/metabolism* ; Neural Stem Cells/metabolism* ; Neurons/metabolism* ; Porphyromonas gingivalis/metabolism* ; Rats ; Rats, Sprague-Dawley ; Tail/metabolism*

OBJECTIVETo observe the time course of proliferation and differentiation of neural stem cells (NSCs) in the subventricular zone (SVZ) of rats following traumatic craniocerebral injury (TBI).

METHODSForty-eight SD rats were randomized into 3 groups, namely the control group without any treatment, the sham-operated group with scalp incision and preparation of a cranial window, and TBI group with craniocerebral injury induced by Feeney's method. With nestin and BrdU as two cell markers, NSE as the neuron-specific marker and GFAP as the glial cell marker, immunofluorescence assay with double labeled antibodies was performed to examine the proliferation and differentiation of endogenous NSCs in the SVZ at different time points after TBI.

RESULTSs The numbers of cells positive for nestin/NSE, nestin/GFAP, BrdU/NSE, and BrdU/GFAP in the SVZ of the rats increased significantly after TBI. The positive cells began to increase at 1 day after TBI, reached the peak level at day 3 and became normal at day 14, showing significant differences between the time points of measurement following TBI and from the cell numbers in the control group measured at the same time points. The cells positive for nestin/ GFAP showed the most distinct increase in the SVZ of the rats with TBI.

CONCLUSIONTBI results in mobilization of the NSCs in the SVZ on the injured side to cause the proliferation and differentiation of the endogenous NSCs. The SVZ is one of the most important germinal centers of NSC proliferation and differentiation.

Animals ; Bromodeoxyuridine ; metabolism ; Cell Differentiation ; Cell Proliferation ; Craniocerebral Trauma ; pathology ; Glial Fibrillary Acidic Protein ; metabolism ; Lateral Ventricles ; cytology ; Nestin ; metabolism ; Neural Stem Cells ; cytology ; Neuroglia ; cytology ; Neurons ; cytology ; Phosphopyruvate Hydratase ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the clinicopathologic features of tuberous sclerosis complex (TSC).

METHODSThe clinicopathologic data of the patients diagnosed as TSC with refractory epilepsy and resection of epileptic focus were retrospectively analyzed.

RESULTSFourteen cases were included, the mean age was (15.8±12.9) years, with a male predominance (male to female ratio=10:4). Frontal lobe was the most common (13/14) site of involvement. MRI showed multiple patchy long T1 and long T2 signals. CT images showed multiple subependymal high density calcified nodules in nine cases. Histology showed mild to severe disruption of the cortical lamination, cortical and subcortical tubers with giant cells and/or dysmorphic neurons. The giant cells showed strong immunoreactivity for vimentin and nestin, while the dysmorphic neurons partially expressed MAP2 and NF. Vimentin also stained strongly the "reactive" astrocytes. Thirteen cases had follow-up information: Engel class I in six cases, Engel class II in six cases, and Engel class III in one case.

CONCLUSIONSDiagnosis of TSC relies on combined pathologic, clinical and neuroradiological features. Immunohistochemical staining can be helpful. Resection of epileptic focus is an effective method to treat refractory epilepsy in TSC.

Adolescent ; Astrocytes ; chemistry ; pathology ; Child ; Drug Resistant Epilepsy ; surgery ; Epilepsy ; complications ; metabolism ; pathology ; Epilepsy, Frontal Lobe ; complications ; metabolism ; pathology ; Female ; Giant Cells ; chemistry ; pathology ; Humans ; Magnetic Resonance Imaging ; Male ; Nestin ; analysis ; Neurons ; metabolism ; pathology ; Retrospective Studies ; Tuberous Sclerosis ; complications ; metabolism ; pathology ; Vimentin ; analysis

Living organisms are exposed to the geomagnetic field (GMF) throughout their lifespan. Elimination of the GMF, resulting in a hypogeomagnetic field (HMF), leads to central nervous system dysfunction and abnormal development in animals. However, the cellular mechanisms underlying these effects have not been identified so far. Here, we show that exposure to an HMF (<200 nT), produced by a magnetic field shielding chamber, promotes the proliferation of neural progenitor/stem cells (NPCs/NSCs) from C57BL/6 mice. Following seven-day HMF-exposure, the primary neurospheres (NSs) were significantly larger in size, and twice more NPCs/NSCs were harvested from neonatal NSs, when compared to the GMF controls. The self-renewal capacity and multipotency of the NSs were maintained, as HMF-exposed NSs were positive for NSC markers (Nestin and Sox2), and could differentiate into neurons and astrocyte/glial cells and be passaged continuously. In addition, adult mice exposed to the HMF for one month were observed to have a greater number of proliferative cells in the subventricular zone. These findings indicate that continuous HMF-exposure increases the proliferation of NPCs/NSCs, in vitro and in vivo. HMF-disturbed NPCs/NSCs production probably affects brain development and function, which provides a novel clue for elucidating the cellular mechanisms of the bio-HMF response.
Animals ; Cell Proliferation ; physiology ; Female ; Magnetic Fields ; Male ; Mice ; Nestin ; metabolism ; Neural Stem Cells ; cytology ; metabolism ; SOXB1 Transcription Factors ; metabolism

OBJECTIVETo explore the nerve regeneration mechanism of electroacupuncture (EA) combined with polysaccharide of gastrodia elate blume (PGB) for secondary thalamic damage of focal cerebral ischemia.

METHODSForty Sprague-Dawley adult rats were randomly divided into a normal control group, a model group, an EA group, a PGB group and an EA + PGB group, 8 rats in each group. The rat model of right middle cerebral artery occlusion was prepared by suture-occluded method. Two weeks after model establishment, rats in the normal control group and model group received no treatment; rats in the EA group were treated with EA at "Baihui" (GV 20) and left "Zusanli" (ST 36), 30 min per treatment, once a day for 14 successive days; rats in the PGB group were treated with intragastric administration of PGB (100 mg/kg) , once a day for 14 days; rats in the EA + PGB group were treated with EA and PGB treatment, once a day for totally 14 days. The expressions of nestin and stem cell factor (SCF) in thalamic ventroposterolateral nucleus (VPL) were detected by immunohistochemical method.

RESULTSThere were positive cells of nestin in ischemia VPL in the model group, and the number of SCF positive cells was increased compared with that in the normal control group (P<0.05). The number of positive cells of nestin and SCF in ischemia VPL in the EA group, PGB group and the EA + PGB group was increased compared with that in the model group (all P<0.05), and the average gray value of immune positive product was all reduced (all P<0.05). The number of positive cells of nestin and SCF in the EA + PGB group was higher than that in the EA group or the PGB group (all P<0.05).

CONCLUSIONEA combined with PGB can significantly increase the SCF expression in ischemia VPL and promote the proliferation of neural stem cells, which is likely to be one of the nerve regeneration mechanism of acupuncture and medication tor secondary thalamic damage of local cerebral isctemia.

Animals ; Brain Ischemia ; drug therapy ; genetics ; metabolism ; therapy ; Combined Modality Therapy ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Electroacupuncture ; Gastrodia ; chemistry ; Humans ; Male ; Nestin ; genetics ; metabolism ; Polysaccharides ; administration & dosage ; Rats ; Rats, Sprague-Dawley ; Stem Cell Factor ; genetics ; metabolism ; Thalamic Nuclei ; metabolism

OBJECTIVETo explore the role of microtubule-associated protein 2 (MAP-2) and nestin in the development of tongue muscles of human embryos and fetuses.

METHODSPV immunohistochemistry was used to detect the expressions of MAP-2 and nestin proteins in the tongue tissues of human embryos and fetuses at the second, third and fourth months of gestation.

RESULTSMAP-2 and nestin positivity was detected in the tongue muscles of human embryos at 2 to 4 months of gestation. In the embryos at the second month of gestation, no obvious MAP-2 positive cells were found in the tongue muscles; at 3 and 4 months, the number of MAP-2-positive cells in the tongue muscles was 24.14∓8.28 and 15.86∓3.89, with the expression intensity of 109.42∓11.62 and 124.27∓8.73, respectively. At 2, 3 and 4 months of gestation, the number of nestin-positive cells in the tongue muscles was 12.50∓3.17, 19.00∓7.63, and 22.80∓6.91, with expression intensity of 119.99∓24.02, 102.20∓11.76, and 98.24∓10.66, respectively. As the gestational age increased, the number of MAP-2-positive cell number continued to decline following a transient increase but the expression intensity kept increasing; nestin-positive cells increased continuously but the expression intensity kept decreasing in the embryonic or fetal tongue muscles.

CONCLUSIONMAP-2 and nestin proteins are involved in the regulation of the development of tongue muscles in human embryos and fetuses.

Humans ; Microtubule-Associated Proteins ; metabolism ; Muscle, Skeletal ; embryology ; Nestin ; metabolism ; Tongue ; embryology

The present paper is aimedto investigate the effect of basic fibroblast growth factor (bFGF) on proliferation, migration and differentiation of endogenous neural stem cell in rat cerebral cortex with global brain ischemia-reperfusion. A global brain ischemia-reperfusion model was established. Immunohistochemistry was used to observe the pathological changes and the expression of BrdU and Nestin in cerebral cortex. RT-PCR was used to measure the NSE mRNA in brain tissue. The results of measurements indicated that in sham operation group, there was no positive cell in cerebral cortex, and the content of NSE mRNA did not change. In the operation group, the expression of BrdU and Nestin increased significantly at the end of the 3rd day, and peaked on the 7th day. NSE mRNA expression did not significantly increase. In bFGF group, compared with sham operation group and model group, the number of BrdU-positive and Nestin-positive cells increased significantly at each time point (P<0. 05), and peaked at the end of the 11th day, and the content of NSE mRNA increased significantly (P<0. 05). This research demonstrated that the proliferation of endogenous neural stem cells in situ could be induced by global cerebral ischemia and reperfu- sion, and could be promoted and extended by bFGF. In additiion, bFGF might promote endogenous neural stem cells differentiated into neurons.
Animals ; Brain Ischemia ; pathology ; Cell Differentiation ; Cell Movement ; Cell Proliferation ; Cerebral Cortex ; cytology ; metabolism ; pathology ; Fibroblast Growth Factor 2 ; pharmacology ; Nestin ; metabolism ; Neural Stem Cells ; drug effects ; Rats ; Reperfusion Injury

OBJECTIVETo establish red-green dual-color fluorescence glioma model in nude mice and to explore its practical values.

METHODSCM-DiI-stained rat glioma C6 cells (C6-CM- DiI cells) expressing red fluorescence were inoculated into the brain of athymic nude mice expressing green fluorescence protein (NC-C57BL/6J-EGFP). Then the whole-body dual-color fluorescence imaging was detected dynamically. Finally whole brains of the tumor-bearing mice were removed and 5 µm thick serial frozen slices were made. Light microscopy, fluorescence microscopy and confocal laser scanning microscopy were performed to observe the transplanted tumor tissue structure and fluorescent cells.

RESULTSTumor mass with red fluorescence increased gradually under continuous in-vivo fluorescence imaging monitoring. Under the fluorescence microscope, cells with red, green and yellow fluorescence were observed in the frozen sections of transplanted tumor tissue and the mutual structural relationship among them could be defined. The tumor cells migration, implantation and cell fusion between transplanted tumor cells and host cells could be observed. It could be distinguished according to the fluorescence, that blood vessels of tumor-origin displayed red fluorescence, blood vessels of host-origin displayed green fluorescence and mosaic blood vessels appeared yellow fluorescence. It was depicted that host innate astrocytes and oligodendrocytes in the microenvironment at the tumor periphery could be activated and dedifferentiated into nestin-positive cells.

CONCLUSIONSIn contrast to traditional animal model, the dual-color fluorescence imaging of nude mouse models of glioma possesses enormous advantages in investigating tumor mass in-vivo fluorescence imaging, tumor cells migration and metastasis, tumor angiogenesis and reactive activation of host innate cells in the microenvironment at tumor periphery, thus, has highly practical application value.

Animals ; Astrocytes ; metabolism ; Brain Neoplasms ; blood supply ; metabolism ; pathology ; ultrastructure ; Carbocyanines ; metabolism ; Cell Fusion ; Cell Line, Tumor ; Cell Movement ; Disease Models, Animal ; Fluorescent Dyes ; metabolism ; Glioma ; blood supply ; metabolism ; pathology ; ultrastructure ; Green Fluorescent Proteins ; metabolism ; Luminescent Proteins ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Nude ; Microscopy, Confocal ; Microscopy, Fluorescence ; Neoplasm Transplantation ; Neovascularization, Pathologic ; Nestin ; metabolism ; Oligodendroglia ; metabolism ; Rats

Astrocytoma ; metabolism ; Glial Fibrillary Acidic Protein ; metabolism ; Humans ; In Vitro Techniques ; Nestin ; metabolism

With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry(+) NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.
Animals ; Cell Differentiation ; Cell Line ; Embryonic Stem Cells ; cytology ; metabolism ; transplantation ; Gene Knock-In Techniques ; Genes, Reporter ; Homologous Recombination ; Humans ; Luminescent Proteins ; genetics ; Mice ; Mice, SCID ; Nestin ; genetics ; Neural Stem Cells ; cytology ; metabolism ; Neurons ; cytology ; metabolism ; Teratoma ; pathology