BACKGROUNDMany researchers suggest that adult mammalian central nervous system (CNS) is incapable of completing self-repair or regeneration. And there are accumulating lines of evidence which suggest that endogenous neural stem cells (NSCs) are activated in many pathological conditions, including stroke in the past decades, which might partly account for rehabilitation afterwards. In this study, we investigated whether there was endogenous neural stem cell activation in intracerebral hemorrhagic (ICH) rat brains.

METHODSAfter ICH induction by stereotactical injection of collagenase type VII into globus pallidus, 5-Bromo-2 Deoxyuridine (BrdU) was administered intraperitoneally to label newborn cells. Immunohistochemical method was used to detect Nestin, a marker for neural stem cells, and BrdU.

RESULTSNestin-positive or BrdU-Labeled cells were predominantly located at 2 sites: basal ganglion around hemotoma, ependyma and nearby subventricular zone (SVZ). No positive cells for the 2 markers were found in the 2 sites of normal control group and sham group, as well as in non-leisioned parenchyma, both hippocampi and olfactory bulbs in the 4 groups. Nestin+ cells presented 4 types of morphology, and BrdU+ nucleus were polymorphologic. Positive cell counting around hemotoma showed that at day 2, Nestin+ cells were seen around hemotoma in model group, the number of which increased at day 4, day 7 (P <0.01), peaked at day 14 (P <0.05), and reduced significantly by day 28 (P <0.01).

CONCLUSIONEndogenous neural stem cells were activated in experimental intracerebral hemorrhagic rat brains.

Animals ; Brain ; pathology ; Bromodeoxyuridine ; metabolism ; Cerebral Hemorrhage ; pathology ; Intermediate Filament Proteins ; analysis ; Male ; Nerve Tissue Proteins ; analysis ; Nestin ; Neurons ; pathology ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; pathology

Animals ; Desmin ; analysis ; genetics ; Female ; Immunohistochemistry ; Intermediate Filament Proteins ; analysis ; genetics ; Kidney Glomerulus ; chemistry ; Nephrosis ; metabolism ; Nerve Tissue Proteins ; analysis ; genetics ; Nestin ; Podocytes ; chemistry ; RNA, Messenger ; analysis ; Rats ; Rats, Inbred WKY ; Vimentin ; analysis ; genetics

OBJECTIVEThis study investigated the effect of hyperbaric oxygenation (HBO) on neural stem cells (NSCs) and myelin in neonatal rats following hypoxic-ischemic brain damage (HIBD) and aimed to explore the possible mechanism of the protective effect of HBO on HIBD.

METHODSSeven-day-old Sprague-Dawley rat pups were randomly assigned into 4 groups: Normal control, HIBD, hyperbaric air (HBA), and HBO groups (n=30 each). The HIBD model was produced by permanent occlusion of the left common carotid artery and 2 hrs hypoxemia exposure (8% O2 at 37 degrees C). HBA and HBO treatment was administered (2 ATA, once daily for 7 days) in the HBA and HBO groups respectively 1 hr after HIBD. BrdU immunohistochemistry was used to detect the NSCs in the sub-ventricle zone (SVZ) of the lateral ventricle and the dentate gyrus (DG) of the hippocampus. The myelin damage was assessed by myelin basic protein (MBP) immunostaining.

RESULTSThe BrdU-positive cells in the SVZ and the DG of the ischemic hemisphere in the HIBD group were dramatically decreased compared with those of the Normal control group at 3 weeks post-HIBD (P < 0.01). The HBO treatment resulted in an increase of BrdU-positive cells in the DG from 153.7 +/- 37.0 to 193.7 +/- 38.8 (P < 0.05). The nestin expression in the HIBD and HBA groups was reduced compared with that in the Normal control group. There was no difference in the nestin expression between the HBO and the Normal control groups. Hypoxia-ischemia (HI) led to marked myelin damage at 1 week post-HIBD. HBO or HBA treatment alleviated the damage.

CONCLUSIONSThe HBO treatment can result in the proliferation of BrdU-positive cells and alleviate the myelin damage following HIBD in neonatal rats, thereby offering neuroprotectivity against HI insults.

Animals ; Animals, Newborn ; Bromodeoxyuridine ; metabolism ; Female ; Hyperbaric Oxygenation ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; therapy ; Immunohistochemistry ; Intermediate Filament Proteins ; analysis ; Male ; Mice ; Myelin Basic Protein ; analysis ; Nerve Tissue Proteins ; analysis ; Nestin ; Neurons ; cytology ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology

OBJECTIVEPrevious studies suggest that hyperbaric oxygen (HBO) treatment promotes the proliferation of neurocytes in neonatal rats following hypoxic-ischemic brain damage (HIBD). The Wnt signaling pathway is associated with neurogenesis. This study examined whether HBO promoted neural stem cells (NSCs) proliferation after HIBD, and whether that the proliferation correlated with Wnt-3 protein expression.

METHODSSeven-day-old Sprague-Dawley rats were randomly divided into three groups: normal control, hypoxia-ischemia (HI), and HI-HBO. HI was induced by the ligation of left common carotid artery, followed by a 2-hr exposure to 8% O2 in the latter two groups. HBO was administered 3 hrs after HI in the HI-HBO group for continuous 7 days (2 atmospheres absolute, once daily). The proliferating NSCs in the subventricular zone (SVZ) was examined by BrdU/nestin immunofluorescence and the expression of Wnt-3 protein in NSCs was examined by nestin/Wnt-3 immunofluorescence at 6 and 24 hrs and at 3, 7 and 14 days of HI. The cellular expressions of nestin and Wnt-3 protein were analyzed by laser scanning confocal microscopy. The linear regression analysis was used to evaluate the correlation between cellular Wnt-3 and nestin protein. The expressions of nestin and Wnt-3 protein in the ischemic cerebral hemisphere were analyzed with Western blotting.

RESULTSThe number of BrdU/nestin positive cells in the SVZ increased 3 hrs after HBO therapy, peaked at 7 days and remained at a higher level until 14 days after HBO therapy in the HI-HBO group compared with the normal control and the HIBD groups. The level of Wnt-3 protein in NSCs increased significantly 3 hrs after HBO therapy, peaked at 3 days and remained at high levels until 14 days after HBO therapy in the HI-HBO group compared with the normal control and the HIBD groups. The level of cellular nestin protein was closely correlated with the level of cellular Wnt-3 protein (r = 0.893, P < 0.05). The Western blotting analysis demonstrated increased Wnt-3 and nestin protein expressions in the ischemic cerebral hemispheres.

CONCLUSIONSHBO treatment promotes the proliferation of NSCs in HIBD neonatal rats, which is correlated with the activation of Wnt signaling.

Animals ; Animals, Newborn ; Blotting, Western ; Bromodeoxyuridine ; metabolism ; Cell Proliferation ; Female ; Fluorescent Antibody Technique ; Hyperbaric Oxygenation ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; therapy ; Intermediate Filament Proteins ; Male ; Nerve Tissue Proteins ; Nestin ; Neurons ; cytology ; Rats ; Stem Cells ; cytology ; Wnt Proteins ; analysis ; Wnt3 Protein

To understand the pathogenesis of proliferative vitreoretinal membrane formation which occurs in proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), etc., accurate identification of the cellular components of the membrane is needed. This study was performed to identify cellular components of the membranes by means of immunohistochemical technique. 11 proliferative vitreoretinal membranes which were surgically obtained from 7 eyes with PVR and 4 eyes with PDR were stained with monoclonal antibodies against cytokeratin, glial fibrillary acidic protein (GFAP), or vimentin using immunoperoxidase technique (ABC method). In the PVR membranes, mean cell positivities for cytokeratin, GFAP and vimentin were 48%, 1% and 92%, respectively and in the PDR membranes, 0%, 5% and 93%, respectively. The above results suggest that retinal pigment epithelial cells and fibroblasts are major cellular components of PVR membranes, and that mesenchymal cells are major cellular components and glial cells are minor cellular components of PDR membranes.
Adolescent ; Adult ; Antibodies, Monoclonal ; Cell Membrane/metabolism/pathology ; Diabetic Retinopathy/metabolism/pathology ; Eye Diseases/metabolism/pathology ; Female ; Humans ; Immunoenzyme Techniques ; Intermediate Filament Proteins/*analysis ; Male ; Middle Aged ; Retinal Diseases/metabolism/*pathology ; Vitreous Body/metabolism/*pathology

To investigate the effect of forced running in motor-driven wheel on neurogenesis in the hippocampal dentate gyrus (DG) of adult rats, 5-bromo-2-deoxyuridine (BrdU), a thymidine analog was applied to mark cell proliferation. Neuroepthelial stem cell protein (nestin) expression was used to identify neural stem/precursor cells. The BrdU- and nestin-positive cells were examined by immunohistochemical technique. The ability of learning was evaluated by Y-maze test to explore the functional role of the newborn cells in the DG after forced running. It was found that the number of BrdU- and nestin-positive cells in the DG in running groups was significantly increased compared to that in the control group (P<0.05). The effect of forced running on neurogenesis was intensity-dependent. In addition, an improvement of learning ability in Y-maze test was observed after forced running. These findings suggest that forced running in motor-driven wheel could enhance neurogenesis in the hippocampal DG of adult rats and improve learning ability.
Animals ; Bromodeoxyuridine ; metabolism ; Cell Survival ; Dentate Gyrus ; cytology ; physiology ; Intermediate Filament Proteins ; analysis ; Learning ; Male ; Maze Learning ; Nerve Tissue Proteins ; analysis ; Nestin ; Neurons ; physiology ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley ; Running

OBJECTIVEBasic fibral growth factor (bFGF) might have a role in the restoration and regeneration of injured neurons following hypoxic-ischemic brain damage (HIBD), but its mechanism has not been fully elucidated. Nestin is an intermediate filament protein expressed in dividing cells during the early stages of CNS development, but it can be reinduced in adults during regeneration of injured neurons after CNS injury. This study investigated the effect of exogenous bFGF on nestin expression in neonatal rats following hypoxia-ischemia (HI) and to explore the possible mechanism.

METHODSEighty-four 7-day-old SD rats were randomly assigned into a Sham-operation group, a HIBD group and a bFGF intervention group (n=28 each). HIBD was induced by ligation of the left carotid artery along with 8% oxygen exposure in neonatal rats from the latter two groups. The Sham-operation group was not subjected to HI. The bFGF intervention group received an intraperitoneal injection of bFGF daily (4000 U/kg). Each group was randomly subdivided into groups sacrificed immediately, at 3, 12 and 24 hrs and 3, 7 and 14 days after HI (n=4). The expression of nestin in the cerebral cortex, hippocampus and extraventricular zone was examined with immunohistochemical staining and image quantitative analysis.

RESULTSNestin was weakly expressed in the hippocampus and extraventricular zone and not expressed in the cortex in the Sham-operation group. The nestin in the cortex, hippocampus and extraventricular zone was significantly increased after HIBD, peaking at 7 days. bFGF treatment increased the nestin expression in the cortex, hippocampus and extraventricular zone and statistical differences were observed from 1 to 14 days after HI when compared with the untreated HIBD group.

CONCLUSIONSExogenous bFGF can up-regulate the nestin expression in neonatal rats following HIBD. The effects of restoration and regeneration of bFGF on injured neurons may be associated with increased nestin expression in neonatal rats.

Animals ; Animals, Newborn ; Female ; Fibroblast Growth Factor 2 ; pharmacology ; therapeutic use ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; pathology ; Immunohistochemistry ; Intermediate Filament Proteins ; analysis ; Male ; Nerve Tissue Proteins ; analysis ; Nestin ; Rats ; Rats, Sprague-Dawley

Research of the FLG mutation in various ethnic groups revealed non-overlapping mutation patterns. In addition, Japanese and Chinese atopic patients showed somewhat different mutations. These ethnic differences make the research on Korean patients mandatory; however, no systematic research on Korean atopic dermatitis (AD) patients has been performed. This study aims to investigate the genetic polymorphism of FLG in Korean atopic dermatitis patients. The study was made up of three groups including 9 Ichthyosis vulgaris (IV) patients, 50 AD patients and 55 normal controls: the ichthyosis group was incorporated due to the reported association between the FLG mutation and IV. In comparison to other sequencing methods, the overlapping long-range PCR was used. We revealed the genetic polymorphism of filaggrin in Koreans, and at the same time, we discovered nonsense mutations in p.Y1767X and p.K4022X in Korean AD patients. By using FLG sequencing techniques confirmed in this study, new mutations or genetic polymorphisms with ethnic characteristics would be detected and further larger studies of repeat number polymorphisms could be performed.
Adult ; Alleles ; Asian Continental Ancestry Group/*genetics ; Base Sequence ; Codon, Nonsense ; DNA/blood/chemistry/metabolism ; DNA Mutational Analysis ; Dermatitis, Atopic/*genetics ; Female ; Genotype ; Heterozygote ; Humans ; Ichthyosis Vulgaris/genetics ; Intermediate Filament Proteins/*genetics ; Male ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide

Epidermal keratinocyte differentiation is a tightly regulated stepwise process that requires protein kinase C (PKC) activation. Studies on cultured mouse keraitnocytes induced to differentiate with Ca2+ have indirectly implicated the involvement of PKC alpha isoform. When PKC alpha was overexpressed in undifferentiated keratinocytes using adenoviral system, expressions of differentiation markers such as loricrin, filaggrin, keratin 1 (MK1) and keratin 10 (MK10) were increased, and ERK1/2 phosphorylation was concurrently induced without change of other MAPK such as p38 MAPK and JNK1/2. Similarly, transfection of PKC alphakinase active mutant (PKC alpha- CAT) in the undifferentiated keratinocyte, but not PKC beta-CAT, also increased differentiation marker expressions. On the other hand, PKC alphadominant negative mutant (PKC beta-KR) reduced Ca2+ -mediated differentiation marker expressions, while PKC beta-KR did not, suggesting that PKC alphais responsible for keratinocyte differentiation. When downstream pathway of PKC alphain Ca2+ - mediated differentiation was examined, ERK1/2, p38 MAPK and JNK1/2 phosphorylations were increased by Ca2+ shift. Treatment of keratinocytes with PD98059, MEK inhibitor, and SB20358, p38 MAPK inhibitor, before Ca2+ shift induced morphological changes and reduced expressions of differentiation markers, but treatment with SP60012, JNK1/2 inhibitor, did not change at all. Dominant negative mutants of ERK1/2 and p38 MAPK also inhibited the expressions of differentiation marker expressions in Ca2+ shifted cells. The above results indicate that both ERK1/2 and p38 MAPK may be involved in Ca2+- mediated differentiation, and that only ERK1/2 pathway is specific for PKCa-mediated differentiation in mouse keratinocytes.
Animals ; Calcium/pharmacology/physiology ; Cell Differentiation/physiology ; Intermediate Filament Proteins/analysis/metabolism ; Keratinocytes/cytology/*enzymology ; Membrane Proteins/analysis/metabolism ; Mice ; Mitogen-Activated Protein Kinase 1/*metabolism ; Mitogen-Activated Protein Kinase 3/*metabolism ; Phosphorylation ; Protein Kinase C/genetics/*physiology ; Research Support, Non-U.S. Gov't ; p38 Mitogen-Activated Protein Kinases/metabolism

OBJECTIVETo explore the way to induce mesenchymal stem cells (MSCs) to differentiate into dopaminergic neurons in vitro.

METHODSMSCs were obtained from rat bone marrow, cultured and passaged. MSCs used in this experiment had multipotency, which was indirectly proved by being induced to differentiate into chondrocytes and adipocytes. MSCs were cultured in medium containing 0.5 mmol/L IBMX for 2 days. Then the medium was replaced with induction medium, which contained GDNF, IL-1beta, mesencephalic glial-cell-conditioned medium and flash-frozen mesencephalic membrane fragments. The surface markers of the differentiated neurons, such as NSE, nestin, MAP-2a, b and TH were detected by immunocytochemistry and Western blot after MSCs were cultured in induction medium for 7 days and 15 days.

RESULTSMSCs differentiated into neural progenitors and expressed nestin after MSCs were incubated with medium containing IBMX for 2 d. After the medium was replaced with induction medium containing many inducing agents, MSCs differentiated into neuron-like cells and dopaminergic neuron-like cells and expressed NSE, MAP-2a, b and TH. The percentage of NSE-positive cells, MAP-2a, b-positive cells and TH-positive cells was 30.032 +/- 2.489%, 41.580 +/- 5.101% and 34.958 +/- 5.534%, respectively after MSCs were induced in medium containing GDNF, IL-1beta, mesencephalic glial-cell-conditioned medium and flash-frozen mesencephalic membrane fragments for 15 days.

CONCLUSIONMSCs can differentiate into dopaminergic neuron-like cells and are a new cell source for the treatment of neurodegeneration diseases and have a great potential for wide application.

Adipocytes ; cytology ; Animals ; Blotting, Western ; Bone Marrow Cells ; Carboxylesterase ; analysis ; Cell Differentiation ; Cells, Cultured ; Chondrocytes ; cytology ; Culture Media, Conditioned ; Dopamine ; analysis ; Intermediate Filament Proteins ; analysis ; Mesencephalon ; cytology ; Mesenchymal Stromal Cells ; cytology ; Nerve Tissue Proteins ; analysis ; Nestin ; Neurons ; cytology ; metabolism ; Phosphoprotein Phosphatases ; analysis ; Rats ; Rats, Wistar