Nestin Protein Expression Research Articles

Investigation of Nestin protein expression in Iraqi patients with high-grade Glioma

Investigation of Nestin protein expression in Iraqi patients with high-grade Glioma

Tetramethylpyrazine inhibits keratitis and neovascularization induced by corneal alkali burn by suppressing the TLR4/NF-κB pathway activation and NLRP1/NLRP3 inflammasomes in rats

The role and related mechanisms of tetramethylpyrazine (TMP) in corneal alkali burn in rats were expected to be explored in this article. After construction of corneal alkali burn rat models, TMP eye drops were given four times daily for consecutive 7 days. H&E staining was utilized for observing the histopathological changes of corneas on the 3rd and 7th days of treatment; immunohistochemistry for detecting the Nestin protein expression changes; qRT-PCR for determining the expression changes of genes correlated with neovascularization [C-X-C Motif Chemokine Ligand 1 (CXCL-1), vascular endothelial growth factor A (VEGFA) and CD31] and inflammation-related factors [monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), tumour necrosis factor α (TNF-α), and IL-6]; Western blot for testing NLR Family Pyrin Domain Containing 1 (NLRP1)/NLRP3 inflammasomes and toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) pathway-related protein expression changes. All above trials were completed based on rat corneal tissue. TMP ameliorated the pathological damage in alkali-burned rat corneal tissue. Specifically, TMP treatment decreased Nestin-positive cell expression and the CXCL-1, VEGFA and CD31 mRNA expression in alkali-burned rat corneal tissue dose-dependently. TMP also down-regulated the IL-1β, TNF-α, MCP-1 and IL-6 mRNA expression and inhibited the NLRP1, caspase-1, NLRP3, pro-IL-1β and mature IL-1β protein expression in the alkali-burned rat corneal tissue. In addition, TMP treatment down-regulated the myeloid differentiation factor 88 (MyD88) and TLR4 protein expression and decreased the p–NF–κB p65/NF-κB p65 ratio in the alkali-burned rat corneal tissue. The mechanism of TMP relieving the inflammatory response and inhibiting neovascularization caused by corneal alkali burn in rats might have a correlation with the suppression of acitivation of TLR4/NF-κB pathway and NLRP1/NLRP3 inflammasomes in rats.

Pre-clinical efficacy evaluation of human umbilical cord mesenchymal stem cells for ischemic stroke.

This study explored the underlying therapeutic mechanism of human umbilical cord mesenchymal stem cells (hUCMSCs) for ischemic stroke (IS), and determined the optimal administration time windows and dose-effect relationship. The levels of SDF-1α, IL-10, IL-6, TNF-α, BDNF, IL-1β, and VEGF-A factors in serum and brain tissue lysate were measured by ELISA. The pathological status of brain tissues was evaluated by Hematoxylin-Eosin (HE) staining, and apoptosis of nerve cells was detected by tunel. The protein expression of CXCR-4, NeuN, and Nestin in the brain tissues was assessed through immunofluorescence. The balance beam, forelimb muscle strength, and limb placement were tested on MCAO rats at different time points and doses. The infarct area of the rat brain tissues was measured at the end of the experiment. The hUCMSC treatment during the acute phase of MCAO significantly reduced the secretion of IL-6, TNF-α, IL-1β but increased IL-10 in serum, and the levels of SDF-α and BDNF in serum and brain tissues lysate were also increased. The pathological results showed that there were more neurons in the treatment group compared to the model group. Immunofluorescence assays showed that the expression of CXCR4、Nestin、NeuN was relatively higher than that in the model group. The d4 and d7 treatment significantly improves the motor function, promotes the recovery of forelimb muscle strength, increases the forelimb placement rate and reduces the scope of cerebral infarction, but the d14 treatment group has less therapeutic effect compared to the d4 and d7 treatment. The 2×107/kg treatment showed the best therapeutic effect, followed by the 1×107/kg treatment, and the worst is 0.5×107/kg treatment from the test of balance beam, forelimb muscle strength, limb placement and the infarct area of the rat brain tissues. The hUCMSCs can inhibit the infiltration of inflammatory cells in the brain tissue, and promote the repair of brain tissue structure and function. Early intervention by injecting high-dose of hUCMSCs can significantly improve the recovery of neurological/motor function and reduce the size of cerebral infarction in rats.

Establishing an Easy-to-Use 3D BM Niche Model for the Co-Culture of Primary Bone Marrow Mesenchymal Stromal Cells (BM-MSC) with Hematopoietic Stem and Progenitor Cells (HSPC)

Background BM-MSC are a collection of primitive cell populations, a major stromal component of the BM niche and are essential to maintain tissue homeostasis. 2D culture protocols are popular, though not standardized, and effective for BM-MSC expansion, but display limited niche function in co-culture systems. 3D co-culture leverages the importance of cell signaling networks activated by cell-cell and cell-matrix interactions in native tissues. Here, we establish a superior and easy-to-use 3D culture protocol and compare to conventional 2D approaches. Methods, Results and Discussion First, we compared commercially available expansion media to the most widely cited investigator-defined media for optimal 2D expansion and 3D culture of ex vivo murine BM-MSC. Based on this, we developed a five-marker flow cytometry panel to accurately quantify BM-MSC culture purity. To test BM-MSC niche function in 2D vs 3D cultures, we generated 3D spheroid hanging drop (HD) cultures from 2D expanded BM-MSC. In HD spheroids, cells are in direct contact with each other as well as extracellular matrix components and can be used to co-culture multiple different cell types. To assess the niche-like function of 3D HD cultures compared to 2D adherent cultures, we assessed Nestin protein expression since Nestin is a marker of HSC niches within mouse BM in vivo. BM-MSC were cultured in 2D or HD and Nestin protein expression assessed by immunofluorescence imaging, showing a significant increase in percent of Nestin-positive cells in HD-cultured BM-MSC, compared to matched 2D controls, in agreement with previous reports in human BM-MSC 3D culture. To assess 3D in vitro BM-MSC niche (IVBMN) function, we assayed stemness markers and survival of FACS isolated primary HSPC (Lineage-Sca1+cKit+, LSK) from mouse BM. LSK cells were cultured as standard (alone) or seeded as co-cultures with BM-MSC within HD with specific cytokine support, in low oxygen (6.5%) conditions (IVBMN) (Figure 1). LSK cells cultured within IVBMN exhibited superior survival as well as retention of stemness markers, demonstrating the benefit of BM-MSC support. In vivo, the BM niche also includes BM niche resident macrophages (BM-Mɸ), which were recently shown to be a limiting factor for engraftment and HSC reconstitution in irradiated mice, and are part of long term and short term HSC niches. To further extend our IVBMN, BM-Mɸ [F4/80+ Ly6G- VCAM1+ CD169+] were collected by FACS and added to IVBMN cultures (IVBMN-Mɸ). Notably, the IVBMN-Mɸ co-culture model further enhanced survival and stemness of primary LSK cells, when compared to LSK cultured alone or within IVBMN. Based on these preliminary data, we demonstrate that our IVBMN-Mɸ culture system is a unique tool which mimics niche properties in vitro and is easy-to-use for HSPC manipulation. Conclusions Here we describe a standardized flow panel for reliable murine BM-MSC immunophenotyping, and characterize a simple but effective 3D BM-MSC co-culture system which allows niche-like function in vitro. Fig. 1. In vitro BM niche (IVBMN) experimental workflow: A) Ex vivo expanded primary BM-MSC (20x) (i) -/+ FACS isolated BM niche resident Mɸ (ii) in co-culture with primary BM HPSC (LSK) (iii). B) Cells were cultured in a 3D mixed culture system with specific cytokine support, in 6.5% low oxygen. C) IVBMN-Mɸ [MSC+ BM-Mɸ +LSK] co-culture promotes maintenance of primary viable LSK compared to IVBMN [MSC+LSK] or LSK standard culture conditions alone, after 5 days culture. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Daily Consumption of High-Polyphenol Olive Oil Enhances Hippocampal Neurogenesis in Old Female Rats

Objective The aim of this study is to evaluate the effect of daily consumption of high-polyphenol (HP) olive oil on neurogenesis by investigating neuronal cell proliferation and maturation in the hippocampus of old rats, and to evaluate the relationship between neurogenesis, spatial memory, and anxiety-like behavior. Methods A total of 34 female, 20–22-month-old Sprague Dawley rats were divided into three groups: control group, low-polyphenol (LP) group, and high-polyphenol (HP) group. The animals were fed distilled water, LP olive oil and HP-extra virgin olive oil, respectively for 6 weeks using an oral gavage. At 43 days, animals were tested using the Morris Water Maze to evaluate spatial memory, and the Open-field test to evaluate anxiety-like behavior. Neural cell proliferation in the dentate gyrus (DG) was determined by BrdU labeling and Nestin protein expression. Neuronal maturation was determined by NeuN labeling. Synaptic density in the hippocampus and prefrontal cortex was examined by measuring Synaptophysin (SYN) levels. Hippocampal Calbindin levels were measured to assess cellular calcium metabolism. Results Daily consumption of HP olive oil significantly improved cell proliferation and neuronal maturation in the DG of old rats. HP-olive oil significantly increased SYN levels in the prefrontal cortex, and nestin and calbindin levels in the hippocampus (p < 0.05). LP olive oil diet has shown no effect on any parameter (p > 0.05). We also did not find any statistically significant difference between the groups in terms of spatial memory and anxiety-like behavior (p > 0.05). Conclusion Our study is first to show that daily consumption of HP-olive oil enhances hippocampal neurogenesis in old rats, which has been confirmed by proliferation and maturation biomarkers. In addition, increased SYN and calbindin levels showed that the generated cells were also functionally developed in the HP group. We suggest that daily consumption of HP olive oil may have beneficial effects on brain aging by triggering neurogenesis.

Repetitive Transcranial Magnetic Stimulation Improves Depression-like Behavior in Rats by Promoting Neural Stem Cell Proliferation and Differentiation

Repetitive transcranial magnetic stimulation (rTMS) is a novel non-invasive neuromodulation technique with neuroprotective properties and is used to treat depression. However, the underlying mechanism of action remains unclear. In this study, we examined the possible mechanism mediating the antidepressant effect of rTMS using animal experiments. Specific pathogen-free rats were treated with rTMS after exposure to social isolation combined with chronic unpredictable mild stress (CUMS). After four weeks of CUMS, the rats exhibited a significant decrease in spatial working memory assessed using open-field testing, a general loss of interest assessed with the sucrose preference test, and a significant reduction in spatial recognition memory ability assessed using the Y-maze. These behavioral deficits were accompanied by decreased numbers of astrocytes in the hippocampus, decreased expression of glial fibrillary acidic protein (GFAP), increased numbers of neural stem cells (NSCs), and increased expression of nestin protein. These results indicated that neuron damage occurred in the depression-like rats. After rTMS intervention, the depression-like behavior was alleviated significantly, and the numbers of NSCs and astrocytes, as well as the expression of GFAP and nestin proteins, returned to normal levels. Overall, it is likely that attenuation of NSC proliferation and differentiation into astrocytes produced a neuroprotective effect on hippocampal neurons, which might partly explain the mechanism by which rTMS alleviates depression.

Investigation of Naoluoxintong on the neural stem cells by facilitating proliferation and differentiation in vitro and on protecting neurons by up-regulating the expression of nestin in MCAO rats

Ethnopharmacological relevanceThe classic traditional Chinese compound Naoluoxintong (NLXT) has been proven an effective remedy for ischemic stroke (IS). The protective effect of NLXT on neural stem cells (NSCs), however, remains unclear. Aim of the studyTo investigate the protective effect of NLXT on NSCs in rats with middle cerebral artery occlusion (MCAO) and the effect of Nestin expression in vivo. Materials and methodsSprague-Dawley (SD) rats were randomly divided into three groups: the sham-operated group, the MCAO model group and the NLXT group. The MCAO model in rats was established by modified Longa wire embolization method. The sham-operated group, the model group and the NLXT groups were divided into three subgroups according to the sampling time points of 1 d, 3 d and 7 d after successful model-making. Immunofluorescence staining, including bromodeoxyuridine (BrdU)/glial fibrillary acidic protein (GFAP), β-tubulinIII/GFAP, BrdU/doublecortin (DCX) and BrdU/neuronal nuclei (NeuN), was used to detect the proliferation and survival of NSCs in the hippocampal after drug administration. Protein expression of Nestin, DCX, GFAP and NeuN in the hippocampal was detected by Western blot (WB). ResultsImmunofluorescence experiment of Nestin labeled: on the first day, a few Nestin-positive cells were found in the hippocampal DG area. Afterwards, the number of Nestin-labeled positive cells in the model group increased, while the number of cells in the sham group did not fluctuate significantly. The number of positive cells in each administration group increased more than that in the model and normal group. β-tubulin III/GFAP double-labeled: a small amount of double labeled cells was expressed in the normal group, and the number subsequently fluctuated little. In the model group, β-tubulin III/GFAP positive cells increased initially after acute ischemia, and gradually decreased afterwards. In the NLXT-treated group, β-Tubulin III positive cells were significantly increased on day 1, 3 and 7, while GFAP positive cells had little change. BrdU/DCX double-labeled: initially, a small number of BrdU/DCX-labeled positive cells were observed in the normal group and the model group, but there was no increasing trend over time. The positive cells in the NLXT group increased over time, and those in the seven-day group were significantly higher than those in the one-day and three-day groups. BrdU/NEUN double-labeled: in the normal group, BrdU/NEUN positive cells were enriched and distributed regularly. The number of positive cells in the model group was small and decreased gradually with time, and the decrease was most obvious on the third day. The number of positive cells in the NLXT group was significantly higher than that in the model group, and the number of positive cells in the seven-day group was significantly higher than that in the one-day and three-day groups. WB results reflected those three proteins, Nestin, NeuN and DCX, showed an increase in expression, except GFAP, which showed a decreasing trend. ConclusionsPreliminarily, NLXT can promote the migration and differentiation of NSCs. It may have a protective effect on the brain by promoting repair of brain tissue damage through upregulation of Nestin after IS.

INVESTIGATION OF THE POTENTIAL EFFECTS OF YAP-1 AND NESTIN ON RISK CLASSES AND PROGNOSIS IN NEUROBLASTOMA

Introduction: Neuroblastoma (NB) is the most common solid tumors in childhood. MYCN is the main prognostic factor for risk classification and staging in NB patients. YAP (Yes Associated Protein) plays a critical role in many types of cancer. In NB, YAP expression levels may be correlate with advanced tumor staging. Nestin is an important neuronal differentiation and cancer stem cell marker in NB. The aim of this study is to examine the potential roles of YAP-1 and Nestin expressions in terms of prognosis and risk classifications for neuroblastoma. Materials and Methods: Histological tumor sections of 55 cases were examined. YAP-1 and Nestin protein expression levels were evaluated by immunohistochemical staining on paraffin sections of different neuroblastoma risk groups. YAP-1 and Nestin expression levels were evaluated with clinical findings of NB patients. P value was &lt;0.05 accepted as a statistically significant. Results: 55 patient tissues were included in this study, of which 24 were male and 21 were female. The ages of the patients are between 1 month to 11 years. YAP-1 protein was expressed in 36.4% in MYCN(+)and 14.6% in MYCN(-) patients. Nestin protein was expressed 36.4% in MYCN(+), and 20.6% in MYCN(-) patients. YAP-1 was expressed 18% in all tissues. Nestin expressions were determined 20.4% in all tissues. YAP-1 was expressed in 26.9% in high-risk and 11.5% in low-risk groups. Nestin was expressed 24.4% in high-risk and 33.3% in low-risk groups. Conclusion: YAP-1 and Nestin might be a clinically risk markers for neuroblastoma patients.

Neural Differentiation Potential of Mesenchymal Stem Cells Enhanced by Biocompatible Chitosan-Gold Nanocomposites.

Chitosan (Chi) is a natural polymer that has been demonstrated to have potential as a promoter of neural regeneration. In this study, Chi was prepared with various amounts (25, 50, and 100 ppm) of gold (Au) nanoparticles for use in in vitro and in vivo assessments. Each as-prepared material was first characterized by UV-visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), scanning electron microscopy (SEM), and Dynamic Light Scattering (DLS). Through the in vitro experiments, Chi combined with 50 ppm of Au nanoparticles demonstrated better biocompatibility. The platelet activation, monocyte conversion, and intracellular ROS generation was remarkably decreased by Chi–Au 50 pm treatment. Furthermore, Chi–Au 50 ppm could facilitate colony formation and strengthen matrix metalloproteinase (MMP) activation in mesenchymal stem cells (MSCs). The lower expression of CD44 in Chi–Au 50 ppm treatment demonstrated that the nanocomposites could enhance the MSCs undergoing differentiation. Chi–Au 50 ppm was discovered to significantly induce the expression of GFAP, β-Tubulin, and nestin protein in MSCs for neural differentiation, which was verified by real-time PCR analysis and immunostaining assays. Additionally, a rat model involving subcutaneous implantation was used to evaluate the superior anti-inflammatory and endothelialization abilities of a Chi–Au 50 ppm treatment. Capsule formation and collagen deposition were decreased. The CD86 expression (M1 macrophage polarization) and leukocyte filtration (CD45) were remarkably reduced as well. In summary, a Chi polymer combined with 50 ppm of Au nanoparticles was proven to enhance the neural differentiation of MSCs and showed potential as a biosafe nanomaterial for neural tissue engineering.

Statins Inhibit the Gliosis of MIO-M1, a Müller Glial Cell Line Induced by TRPV4 Activation.

We characterized Müller cell gliosis induced by the activation of transient receptor potential vanilloid-type 4 (TRPV4) and assessed whether statins could modulate the gliosis. The human Müller cell line, MIO-M1, was used to analyze the gliosis caused by glaucomatous stimulation. To induce Müller gliosis in MIO-M1 cells, GSK101 was used to activate TRPV4, and Müller gliosis was evaluated by analyzing vimentin, nestin, and glial fibrillary acidic protein (GFAP) expression. The expression level of TNF-α was determined by ELISA. To evaluate the GSK101 activation of the NF-κB pathway, p65 phosphorylation was measured by Western blotting, and the nuclear translocation of p65 and IκBα phosphorylation were assessed by immunostaining. To assess the effect of statins on MIO-M1 gliosis, cells were pretreated for 24 h with statins before GSK101 treatment. Vimentin, nestin, and GFAP expression were upregulated by GSK101, while statins effectively inhibited them. The expression of TNF-α was increased by GSK101. The phosphorylation and nuclear translocation of p65 and IκBα phosphorylation, which occurs prior to p65 activation, were induced. Statins suppressed the GSK101-mediated phosphorylation of IκBα and p65 translocation. Statins can mitigate gliosis in the human Müller cell line. Because TRPV4 activation in Müller cells reflects glaucoma pathophysiology, statins may have the potential to prevent RGC death.

Vitamin D Receptor/Vitamin D Response Element Directly Modulate Nestin Transcription to Ameliorate PAN-Induced Podocyte Morphological Changes

Background: The change of podocyte morphology is a pathologic feature of chronic kidney disease. Several studies have suggested that vitamin D plays a role in the protection of podocytes, but the underlying mechanism remains unclear. Methods: The effects of paricalcitol on podocyte injury were tested in a puromycin aminonucleoside (PAN)-induced rat model and cultured mouse podocytes. Proteinuria, podocyte foot process (FP) effacement, and the expression of nestin and vitamin D receptor (VDR) were evaluated. VDR-siRNA or plasmids containing VDR-shRNA were transfected into podocytes to silence VDR expression. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to verify the connection between VDR and nestin gene expression. Results: Paricalcitol significantly alleviated proteinuria and podocyte FP effacement in PAN-induced nephrosis, which was accompanied by increased VDR expression in the glomeruli. Paricalcitol also inhibited PAN-induced nestin overexpression in the glomeruli. In an in vivo study, PAN significantly inhibited VDR protein expression, stimulated nestin protein expression, and resulted in nestin filament derangement in mouse podocytes, while paricalcitol treatment abolished these effects. In contrast, downregulation of VDR resulted in derangement and overexpression of nestin. ChIP assays demonstrated the presence of a vitamin D response element (VDRE) in the nestin promoter, and paricalcitol enhanced the binding of VDR to VDRE. Furthermore, luciferase reporter assays of the nestin promoter fragment showed that paricalcitol effectively repressed nestin reporter gene expression after PAN treatment, and mutation of VDRE abolished this effect. Conclusions: Paricalcitol directly regulates nestin transcription through the interaction of VDR/VDRE, thereby preventing morphological changes of podocytes in PAN nephropathy.

Exploration of the role of the subodontoblastic layer in odontoblast-like cell differentiation after tooth drilling using Nestin-enhanced green fluorescent protein transgenic mice

ObjectivesOriginal odontoblasts and regenerated odontoblast-like cells (OBLCs) may differently regulate Nestin expression. This study aimed to investigate the role of the subodontoblastic layer (SOBL) using green fluorescent protein (GFP) reactivity in the process of OBLC differentiation after tooth drilling in Nestin-enhanced GFP transgenic mice. MethodsA groove-shaped cavity was prepared on the mesial surface of the maxillary first molars of 5- or 6-week-old mice under deep anesthesia. Immunohistochemical staining for Nestin and GFP and Nestin in situ hybridization were conducted on the sections obtained at 1–14 days postoperative. ResultsOdontoblasts showed intense endogenous Nestin protein and mRNA expression, whereas the coronal SOBL cells showed a Nestin-GFP–positive reaction in the control groups. The injured odontoblasts had significantly decreased Nestin immunoreactivity as well as decreased expression of Nestin mRNA 1–2 days after the injury; subsequently, newly differentiated OBLCs were arranged along the pulp–dentin border, with significantly increased Nestin expression as well as increased expression of Nestin mRNA on days 3–5 to form reparative dentin. Nestin-GFP–positive cells at the pulp–dentin border significantly increased in number on days 1 and 2. GFP(+)/Nestin(+) and GFP(−)/Nestin(+) cells were intermingled in the newly differentiated OBLCs. ConclusionsThe commitment of Nestin-GFP–positive cells into Nestin-positive OBLCs suggests that the restriction of endogenous Nestin protein and mRNA expression in the static SOBL cells was removed by exogenous stimuli, resulting in their migration along the pulp–dentin border and their differentiation into OBLCs.

Altered uterine angiogenesis in rats treated with a glyphosate-based herbicide

Glyphosate-based herbicides (GBHs) are the agrochemicals most used around the globe. However, they might have adverse effects on human and animal health. Previously, we showed that female rats neonatally exposed to GBHs exhibit altered expression of morphogenetic molecules and biomarkers of uterine development. We also observed a reduction in the size of implantation sites, altered expression of decidualization-related molecules, and increased post-implantation losses. Since decidualization comprises morphogenetic, biochemical and vascular changes, here we investigated the effects of neonatal GBH exposure on uterine angiogenesis in neonatal and pregnant rats. To achieve this, Wistar female rats were exposed to saline solution or GBH (2 mg glyphosate/kg-bw/day) on post-natal days (PND) 1, 3, 5 and 7. On PND8, uterine samples were collected for developmental studies. On PND90, the remaining females were mated and in the morning of gestational day (GD) 9, the implantation sites were collected. Angiogenesis-related molecules and cells involved in this process were identified and/or measured by immunohistochemistry or RT-PCR. On PND8, GBH-treated rats showed increased vascular endothelial growth factor (VEGF) expression and decreased Notch1, inducible nitric oxide synthase (iNOS) and Angiopoietin-2 (Ang2) mRNA levels. Vascular area, vessel diameter, endothelial cell proliferation, VEGF and Nestin protein expression, and VEGF, Notch1, iNOS and cyclooxygenase-2 (Cox-2) genes were downregulated in implantation sites of exposed females, while Ang2, VEGF receptor 1 and interleukin-10 (IL-10) were increased. Mast cells and macrophages were increased on PND8 and GD9 of treated rats. The increased Transforming growth factor-beta expression in the antimesometrial zone and IL-10 mRNA expression suggest that the M2 type is the predominant population of macrophages on implantation sites. In conclusion, neonatal GBH exposure alters the expression of angiogenesis-related molecules at neonatal uterine development and decidual reaction, suggesting altered vascular support. These alterations might contribute to the increased post-implantation losses observed in GBH-treated rats.

Buyang Huanwu Decoction promotes neurogenesis via sirtuin1/autophagy pathway in a cerebral ischemia model.

Stroke is one of the main causes of disease-related mortality worldwide. Buyang Huanwu Decoction (BHD) has been used to protect against stroke and stroke-induced disability for several years in China. Studies have shown that BHD can relieve neuronal damage in rats with cerebral ischemia/reperfusion (I/R) injury. However, the mechanism remains unclear. A middle cerebral artery occlusion and reperfusion (MCAO-R) model was used in the present study. The animals were treated with BHD (5, 10 and 20 g/kg) or rapamycin. Infarct size and modified neurological severity score were calculated on day 5 following MCAO-R surgery. Cellular changes around the ischemic penumbra were revealed by hematoxylin and eosin and Nissl staining. The protein expression levels of nestin, brain-derived neurotrophic factor (BDNF), doublecortin on the X chromosome (DCX) and autophagy-related proteins (beclin 1, LC3-II and p62) in the peri-ischemic area of the brain were detected. The results demonstrated that post-surgical treatment with BHD reduced the brain infarct size and improved neurological deficits in MCAO-R rats. BHD protected against MCAO-R-induced neuronal impairment and promoted neurogenesis, increased the protein expression of nestin, BDNF and DCX and markedly enhanced autophagy by increasing beclin 1 and LC3-II and decreasing p62. Meanwhile, BHD promoted the expression of sirtuin 1 (SIRT1), an important regulator of autophagy. In conclusion, the present study suggested that post-surgical treatment with BHD could protect rat brains from I/R injury, potentially through the SIRT1/autophagy pathway.

Insulin Promotes Schwann-Like Cell Differentiation of Rat Epidermal Neural Crest Stem Cells.

Schwann cells (SCs) are considered potentially attractive candidates for transplantation therapies in neurodegenerative diseases. However, problems arising from the isolation and expansion of the SCs restrict their clinical applications. Establishing an alternative Schwann-like cell type is a prerequisite. Epidermal neural crest stem cells (EPI-NCSCs) are well studied for their autologous accessibility, along with the ability to produce major neural crest derivatives and neurotrophic factors. In the current study, we explored insulin influence, a well-known growth factor, on directing EPI-NCSCs into the Schwann cell (SC) lineage. EPI-NCSCs were isolated from rat hair bulge explants. The viability of cells treated with a range of insulin concentrations (0.05-100 μg/ml) was defined by MTT assay at 24, 48, and 72 h. The gene expression profiles of neurotrophic factors (BDNF, FGF-2, and IL-6), key regulators involved in the development of SC (EGR-1, SOX-10, c-JUN, GFAP, OCT-6, EGR-2, and MBP), and oligodendrocyte (PDGFR-α and NG-2) were quantified 1 and 9 days post-treatment with 0.05 and 5 μg/ml insulin. Furthermore, the protein expression of nestin (stemness marker), SOX-10, PDGFR-α, and MBP was analyzed following the long-term insulin treatment. Insulin downregulated the early-stage SC differentiation marker (EGR-1) and increased neurotrophins (BDNF and IL-6) and pro-myelinating genes, including OCT-6, SOX-10, EGR-2, and MBP, as well as oligodendrocyte differentiation markers, upon exposure for 9 days. Insulin can promote EPI-NCSC differentiation toward SC lineage and possibly oligodendrocytes. Thus, employing insulin might enhance the EPI-NCSCs efficiency in cell transplantation strategies.

MicroRNA-125b and its downstream Smurf1/KLF2/ATF2 axis as important promoters on neurological function recovery in rats with spinal cord injury.

The purpose of this study is to investigate the role of microRNA‐125b (miR‐125b) and its mechanism in spinal cord injury (SCI) by targeting Smurf1. After loss‐ and gain‐function approaches were conducted in SCI rat models and neural stem cells (NSCs) isolated from foetal rats, the Basso‐Beattie‐Bresnahan (BBB) score was calculated, and related protein expression was determined by Western blot analysis and cell apoptosis by TUNEL staining. NSC viability was detected by CCK‐8, migration abilities by Transwell assay and apoptosis by flow cytometry. The relationship between miR‐125b, Smurf1 and KLF2 was evaluated by dual‐luciferase reporter gene experiments, Co‐IP and in vivo ubiquitin modification assays. Inhibition of miR‐125b and KLF2 and the up‐regulation of Smurf1 and ATF2 were observed in SCI rats. BBB scores were elevated, the expression of Nestin, NeuN, GFAP, NF‐200 and Bcl‐2 protein was enhanced but that of Bax protein was reduced, and cell apoptosis was inhibited in SCI rats after up‐regulating miR‐125b or silencing ATF2. Smurf1 was a target gene of miR‐125b, which promoted KLF2 degradation through its E3 ubiquitin ligase function, and KLF2 repressed the expression of ATF2 in NSCs. The results in vivo were replicated in vitro. miR‐125b overexpression promotes neurological function recovery after SCI.

Polycomb group protein Bmi1 is required for the neuronal differentiation of mouse induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) reprogrammed by somatic cells may be used as a potentially novel treatment regimen in stem cell regenerative medicine, particularly in the central nervous system (CNS). In the present study, iPSCs were generated using mouse embryonic fibroblasts by ectopic overexpression of Sox-2, Oct-3/4, Klf-4 and c-Myc, and cultured under the same conditions as that used for embryonic stem cells. The neuronal differentiation capacity of mouse iPSCs was examined, and the involvement of the formation of embryoid bodies was assessed. The results suggested that after 15 days of neuronal inducement, Nestin, Vimentin and Glast protein expression levels were significantly increased in the mouse iPSC-derived cells. Additionally, Bmi1, which is selectively expressed in differentiated postnatal adult stem cells. such as hematopoietic stem cells and neural stem cells, was required for establishment of the neuronal differentiation of mouse iPSCs. In order to assess the effects of Bmi1 in neuronal differentiation, Bmi1 expression levels were inhibited with the small molecule PTC-209. The results showed that inhibition of Bmi1 expression reduced the expression of neuronal markers, such as Nestin, compared with the controls. These results suggested that mouse iPSCs can be induced to achieve neuronal differentiation. More interestingly, Bmi1 was required during the neuronal differentiation of mouse iPSCs.

Effects of in vitro exposure of perfluorooctanoic acid and monocrotophos on astroglia SVG p12 cells

Glia cells provide supportive functions to the central nervous system and can be compromised by environmental contaminants. The primary objective of this study was to characterize the effects of in vitro exposure to perfluorooctanoic acid, a persistent environmental contaminant and/or monocrotophos (MCP), a neurotoxic organophosphate that is rapidly metabolized, to astroglia SVG p12 cells. The endpoints evaluated include cell viability, intracellular glutamate levels as a marker of astrocyte homeostasis function, differential gene expression for selected proteins, which include inflammatory markers (tachykinin), astrocytosis (nestin), S100B, and metabolism enzymes (CYP1A1). The results from cell viability revealed significant differences from the controls at some of the concentrations tested. Also, intracellular glutamate levels were elevated at the 10-μM concentration for perfluorooctanoic acid (PFOA) as well as the 10-μM PFOA/5-μM MCP concentration. Gene expression results at 80-μM PFOA concentration revealed a significant increase in the expression of S100B, tachykinin and CYP1A1. A combination of 10-μM PFOA/20-μM MCP caused a significant decrease in the expression of tachykinin. Gene expression for MCP exposures produced a decrease at the 20-μM MCP concentration. Immunofluorescence results indicated an increase in nestin protein expression for the 20-μM concentration of MCP, which contradicted the gene expression at the same concentration tested. The results indicate that toxicity to glia cells can compromise critical glia functions and could be implicated in neurodegenerative diseases.

Thyroid Hormone Signaling in Embryonic Stem Cells: Crosstalk with the Retinoic Acid Pathway.

While the role of thyroid hormones (THs) during fetal and postnatal life is well-established, their role at preimplantation and during blastocyst development remains unclear. In this study, we used an embryonic stem cell line isolated from rat (RESC) to study the effects of THs and retinoic acid (RA) on early embryonic development during the pre-implantation stage. The results showed that THs play an important role in the differentiation/maturation processes of cells obtained from embryoid bodies (EB), with thyroid hormone nuclear receptors (TR) (TRα and TRβ), metabolic enzymes (deiodinases 1, 2, 3) and membrane transporters (Monocarboxylate transporters -MCT- 8 and 10) being expressed throughout in vitro differentiation until the Embryoid body (EB) stage. Moreover, thyroid hormone receptor antagonist TR (1-850) impaired RA-induced neuroectodermal lineage specification. This effect was significantly higher when cells were treated with retinoic acid (RA) to induce neuroectodermal lineage, studied through the gene and protein expression of nestin, an undifferentiated progenitor marker from the neuroectoderm lineage, as established by nestin mRNA and protein regulation. These results demonstrate the contribution of the two nuclear receptors, TR and RA, to the process of neuroectoderm maturation of the in vitro model embryonic stem cells obtained from rat.

Expression of Nestin and Glial Fibrillary Acidic Protein in the Marginal Ischemic Zone of the Brain in SHR Rats.

We studied spatial organization and structural characteristics of striatal glial cells in spontaneously hypertensive rats (SHR) in 48 h after 30-min focal ischemia. Immunocytochemical analysis of nestin and glial fibrillar acidic protein (GFAP) revealed 3 types of activated astrocytes: expressing only nestin, only GFAP, or both markers. There were no nestin-immunopositive astrocytes in the striatum of sham-operated rats. In cells expressing nestin and GFAP, localization of these markers did not completely coincide, which can be explained by different functions of these proteins or formation of heterodimers of nestin with other intermediate filament proteins.