Co-localization Of BrdU Research Articles

Leonurine Regulates Hippocampal Nerve Regeneration in Rats with Chronic and Unpredictable Mild Stress by Activating SHH/GLI Signaling Pathway and Restoring Gut Microbiota and Microbial Metabolic Homeostasis.

Depression is a highly prevalent and heterogeneous disorder that requires new strategies to overcome depression. In this study, we aimed to investigate whether leonurine modulated hippocampal nerve regeneration in chronic and unpredictable mild stress (CUMS) rats through the SHH/GLI signaling pathway and restoring gut microbiota and microbial metabolic homeostasis. The CUMS rat model was constructed and treated with leonurine. The body weight of rats was recorded, and a series of tests were performed. Western blot was utilized to measure the expression of BDNF and 5-HT in the hippocampus. Then the expression of SHH, GLI, PTCH, and SMO were measured by qRT-PCR and western blot. The colocalization of BrdU+DCX and BrdU+NeuN was evaluated by IF. 16S rDNA high-throughput sequencing was applied to detect the composition and distribution of gut microbiota. The differential metabolites were analyzed by untargeted metabolomics. The correlation between gut microbiota and microbial metabolites was analyzed by Pearson correlation coefficient. After CUMS modeling, the body weight of rats was decreased, and the expression of BDNF and 5-HT were decreased, while the body weight was recovered, and the expression of BDNF and 5-HT were increased after leonurine treatment. Leonurine reversed the reduction in the colocalization of BrdU+DCX and BrdU+NeuN and the reduction in the levels of SHH, GLI, PTCH, and SMO induced by CUMS modeling. Leonurine also restored gut microbiota and microbial metabolites homeostasis in CUMS rats. Furthermore, Prevotellaceae_Ga6A1_group was negatively correlated with 3-Oxocholic acid, nutriacholic acid, and cholic acid. Collectively, leonurine regulated hippocampal nerve regeneration in CUMS rats by activating the SHH/GLI signaling pathway and restoring gut microbiota and microbial metabolic homeostasis.

Effect of thrombin preconditioning on migration of subventricular zone-derived cells after intracerebral hemorrhage in rats

Objective: To investigate the effect of thrombin preconditioning (TPC) on the intracerebral hemorrhage (ICH)-induced proliferation, migration, and function of subventriclular zone (SVZ) cells and to find new strategies that enhance endogenous neurogenesis after ICH.Methods: Male Sprague-Dawley rats were randomly divided into 3 groups (ICH, TPC, and control group). Rats of each group were randomly divided into 5 subgroups (3-d, 7-d, 14-d, 21-d, and 28-d subgroup). ICH was caused by intrastrial stereotactic administration of collagenase type IV. Brdu was used to label newborn SVZ cells. Organotypic brain slices were cultured to dynamically observe the migration of SVZ cells at living brain tissue. Migration of Dil-labeled SVZ cells in living brain slices was traced by time-lapse microscopy. To assess whether SVZ cells migrating to injured striatum had the ability to form synapses with other cells, brain slices from each group were double immunolabeled with Brdu and synapsin I.Results: The number of Brdu-positive cells markedly increased in the ipsilateral SVZ and striatum 3 days after TPC, peaked at 14 days (P < 0.01), continued to 21 days, and then gradually decreased at 28 days with significant difference compared to the ICH group at each time point (P < 0.01). Migration of Dil-labeled SVZ cells in brain slices in each group was observed and imaged during a 12-h period. Dil-labeled SVZ cells in the TPC group were observed to migrate laterally toward striatum with time with a faster velocity compared to the ICH group (P < 0.01). Our study also demonstrated that TPC induced strong colocalization of Brdu and synapsin I in the ipsilateral striatum between 3 and 28 days after injury.TPC made colocalization of Brdu and synapsin I appear earlier and continue for a longer time compared to the ICH group.Conclusions: Our results demonstrated that TPC could promote proliferation, migration, and function of SVZ cells after ICH, which may provide a new idea for enhancing endogenous neurogenesis and developing new therapeutic strategies against ICH-induced brain injury.

Neural Stem/Progenitor Cells of the Spinal Cord are Activated in Response to Tail Loss in the Leopard Gecko

As for many lizards, the leopard gecko (Eublepharis macularius) is able to self‐detach its tail as an anti‐predation strategy and subsequently regenerate a replacement. The replacement tail includes a regenerated spinal cord. We hypothesize that endogenous neural stem progenitor cells (NSPCs) are present in the original spinal cord and that these cells are activated during regeneration. As many stem cells are slow cycling, we used a Bromodeoxyuridine (BrdU) pulse‐chase experiment to identify label‐retaining populations in the original spinal cord. We also conducted a spatiotemporal characterization of spinal cord cells before and during regeneration using a panel of common NSPC markers (including Sox2, Sox9, Vimentin, βIII‐Tubulin and Musashi‐1, and the proliferation marker PCNA). BrdU retaining cells were still present after a 20‐week chase. Prior to injury, expression of NSPC markers is limited, but co‐localization of BrdU and Sox2 in ependymal cells was observed. Following tail loss, ependymal cells in the original stump of the tail become highly proliferative and express our panel of NSPC markers. Outgrowth of the new tail is matched by proliferating, NSPC marker‐positive ependymal cells. These cells appear to contribute to regenerating spinal cord. We conclude that ependymal cells are a likely source of endogenous NSPCs in the gecko and that they are activated following tail loss.NSERC Grant: 400358

Prenatal Carbofuran Exposure Inhibits Hippocampal Neurogenesis and Causes Learning and Memory Deficits in Offspring

Neurogenesis is a process of generation of new neurons in the hippocampus and associated with learning and memory. Carbofuran, a carbamate pesticide, elicits several neurochemical, neurophysiological, and neurobehavioral deficits. We evaluated whether chronic prenatal oral exposure of carbofuran during gestational days 7-21 alters postnatal hippocampal neurogenesis at postnatal day 21. We found carbofuran treatment significantly decreased bromodeoxyuridine (BrdU) positive cell proliferation and long-term survival in the hippocampus only but not in the cerebellum. We observed a reduced number of transcription factor SOX-2 and glial fibrillary acidic protein (GFAP) colabeled cells, decreased nestin messenger RNA (mRNA) expression, and decreased histone-H3 phosphorylation following carbofuran treatment, suggesting a decreased pool of neural progenitor cells (NPC). Colocalization of BrdU with doublecortin (DCX), neuronal nuclei (NeuN), and GFAP suggested decreased neuronal differentiation and increased glial differentiation by carbofuran. The number of DCX(+) and NeuN(+) neurons, NeuN protein levels, and fibers length of DCX(+) neurons were decreased by carbofuran. Carbofuran caused a significant downregulation of mRNA expression of the neurogenic genes/transcription factors such as neuregulin, neurogenin, and neuroD1 and upregulation of the gliogenic gene Stat3. Carbofuran exposure led to increased BrdU/caspase 3 colabeled cells, an increased number of degenerative neurons and profound deficits in learning and memory processes. The number and size of primary neurospheres derived from the hippocampus of carbofuran-treated rats were decreased. These results suggest that early gestational carbofuran exposure diminishes neurogenesis, reduces the NPC pool, produces neurodegeneration in the hippocampus, and causes cognitive impairments in rat offspring.

Roles of Id1 in Aβ-induced cell cycle re-entry in postmitotic neurons

Senile plaque containing aggregation of amyloid-beta peptide (Aß) is one of the major hallmarks of Alzheimer's disease (AD). Several lines of evidence suggest the correlation between Aß-induced neuronal death and cell cycle re-entry, but the underlying mechanisms remain unclear. Inhibitor of differentiation 1 (Id1) is involved in the regulation of cellular differentiation and cell cycle progression. Doublecortin (DCX) is a neuronal lineage marker that expresses at proliferative stage and early differentiating stage. Subsequent to exit from cell cycle, NeuN is expressed at early and late postmitotic maturation stage. Glial fibrillary acidic protein (GFAP) is a specific protein marker for astrocyte. Bromodeoxyuridine (BrdU) is incorporated during DNA synthesis in proliferating cells. Thus, colocalization of BrdU with DCX or NeuN in fully differentiated neurons may serve as a marker for cell cycle re-entry. We investigate whether Id1 induction by Aß causes cell cycle re-entry in postmitotic neurons leading to cell death. The expression profiles of Id1 and cyclins upon exposure to Aß25-35 were determined by real-time RT-PCR and Western blotting in primary cortical neurons. Immunocytochemistry was utilized to quantitatively determine the numbers of DCX+/BrdU+, NeuN+/BrdU+, GFAP+/BrdU+, and caspase 3+/BrdU+ cells after Aß25-35 treatment. Finally, siRNA was employed to suppress Id1 expression in cortical neurons. Aß25-35 induced Id1, cyclin D1, E1 and A2 mRNA in primary cortical neurons. Aß25-35 and Aß1-42 induced expression of Id1 protein. Aß25-35 increased the numbers of DCX+/BrdU+, NeuN+/BrdU+, GFAP+/BrdU+, and caspase 3+/BrdU+ cells, indicating Aß25-35-mediated interference of neuronal differentiation, increases in astrocytosis, and a heightened extent of apoptosis in cultured cortical neurons. Suppression of Id1 expression by siRNA in part recovered cell survival after Aß25-35 and Aß1-42 treatments in primary cortical neurons. Id1 induction by Aß may play a significant role in Aß-induced cell cycle re-entry and neuronal death.

P86. Identification of urothelial stem cells for tissue engineering

Introduction Stem cells (SCs) are able to self-renew and to differentiate into various cell types. During life, SCs are involved in organogenesis, but also in maintenance and repair of adult tissue. In reconstructive medicine, the availability of transferable tissue often is limited. For reconstructive tissue engineering, the combination of SCs with biomatrices may be a viable alternative approach. Urothelium, a layer of epithelial cells lining the bladder wall, is composed of basal, intermediate, and superficial cells. It has been hypothesized that the SCs are located in the basal cell layer. Since isolating urothelial SCs to combine with biomatrices may generate a basic construct for bladder tissue engineering, the identification and selection of urothelial SCs are crucial issues that need to be investigated. The aim of this study is to identify SCs from urothelium by identifying slow-cycling BrdU-retaining cells together with candidate SCs markers. Method: Tissue from newborn (5 days postnatal), pubertal (1 month), and adult (6 months) mice harvested from BrdU pulsed pregnant mice was examined by immunohistochemistry. Additionally, harvested tissue was examined for cells with a potential SC phenotype, including Cytokeratin 7 (CK7), Cytokeratin 14 (CK14), Cytokeratin 20 (CK20), and β-catenin. Result: The potential SCs in bladder were identified by the BrdU-retained cell population in 1 month old mice of ~8% and 6-month-old mice of ~1%. Most of BrdU retained cells were localized in basal part of epithelium as detected by co-localization of BrdU with CK 7, the latter staining all basal cells. CK14 was differentially expressed in newborn and adult mice, with only rare cells in the adult basal cell layer. In a minority population of BrdU (less than 0.1%) retained cells in 6-month-old mice were located in the umbrella cells (CK 20 positive). β-catenin showed ubiquitous staining of basal urothelium. Conclusion BrdU retained cells were reduced over time in adult life, suggesting a SC pool in the adult bladder. The markers being used showed differential localization and cell lineage of BrdU retained cells, where the basal cell layer contained the lion share of BrdU retained cells. Identification of membrane markers in the BrdU pool may enable cell sorting and isolation of bladder SCs. Ultimately isolated cells can then be expanded and cultured on biomatrices to generate a basic construct for bladder tissue engineering, and to repair and restore bladder function.

Loss of Sonic Hedgehog in the adult stomach results in foveolar hyperplasia and delayed differentiation of the zymogenic cell lineage

ObjectiveParietal cell‐expressed Sonic Hedgehog (Shh) regulates function and differentiation of the gastric epithelium. Correlative evidence shows that loss of Shh expression associated with Helicobacter pylori infection results in the disruption of the normal gastric epithelium. We sought to identify whether Shh regulates gastric epithelial cell differentiation in the absence of inflammation.MethodsA parietal cell‐specific, tamoxifen‐inducible deletion of Shh mouse model was developed (HKCreInd/ShhKO). Epithelial cell proliferation and migration was studied using BrdU and Ki67 co‐immunostaining. Cell lineage changes were studied using markers specific for surface mucous (Ulex europaeus, UEAI), mucous neck (Griffonia simplicifolia, GSII) and zymogen (intrinsic factor, IF) cells.ResultsCompared to Controls, HKCreInd/ShhKO mice developed foveolar hyperplasia reflected by an expansion of BrdU/UEAI positive cells. HKCreInd/ShhKO mice also showed an expansion of GSII/IF co‐expressing cells at the base of the gastric gland. Co‐localization of BrdU and Ki67 revealed loss of normal migration of the epithelial cells within HKCreInd/ShhKO mouse stomachs.ConclusionLoss of Shh in the adult stomach results in the development of foveolar hyperplasia and delayed differentiation of the zymogenic cell lineage independent of inflammation.(DHC Pilot and Feasibility Project Award, Zavros)

Intrinsic neurogenesis in the cerebral cortex continues in adult guinea pig and gives rise to new GABAergic neurons

We have identified neuroblast‐like and immature neurons appearing to be born within the adult guinea pig cerebral cortex. Morphologically diverse cells expressing doublecortin (DCX) were confined to layer II and adjacent borders. Cells (often in clusters) varied in size, shape, label‐intensity, nuclear morphology and with somata appearing in contact with one another. DCX cells co‐expressed polysialylated neural cell adhesion molecule (PSA‐NCAM) and neuron‐specific type III beta‐tubulin. Larger DCX+ cells had well‐developed processes and expressed neuron‐specific nuclear protein (NeuN). Some DCX cells displayed reduced DCX staining but elevated NeuN expression, suggesting a transitional state of maturity. “Transitional” cells also showed immunoreactivities to GABA and GAD67. Cells expressing mitotic markers (proliferating cell nuclear antigen, Ki67, or 5‐bromo‐2′‐deoxyuridine following short survival times, were also encountered across the cortex with prevalence in layers I and II. Co‐localization of BrdU and DCX was seen in several cells following longer survival periods. Our findings suggest the superficial layers of the guinea pig cerebral cortex to be neurogenic throughout life and to provide new GABAergic neurons during adulthood. Supported by Southern Illinois University and Illinois Department of Public Health.

Vasopressin Mediates Mitogenic Responses to Adrenalectomy in the Rat Anterior Pituitary

To determine whether increased vasopressinergic activity during chronic stress or adrenalectomy mediates trophic changes in the corticotroph, we examined the effect of peripheral V1 receptor blockade in rats, using the antagonist, dGly[Phaa1,D-tyr(et), Lys, Arg]vasopressin (VP), on the number of pituitary cells taking up bromodeoxyuridine (BrdU) and cells containing immunoreactive ACTH (irACTH). Adrenalectomy significantly increased the number of BrdU- and ACTH-labeled cells at 3 and 6 d, and a much larger increase was observed at 28 d. Minipump infusion of V1 antagonist for 28 d, at doses blocking the increases in ACTH and corticosterone induced by exogenous VP, prevented the increases in BrdU incorporation, but not irACTH cells observed 28 d after adrenalectomy. Unexpectedly, colocalization of BrdU with ACTH-positive cells was minor (about three cells per pituitary section), and this was unaffected by adrenalectomy or V1 antagonist infusion. In contrast, adrenalectomy for 6 or 14 d failed to increase BrdU incorporation or irACTH cells in V1b receptor knockout mice while inducing the expected increase in wild-type mice. The data show that VP is required for pituitary mitogenesis after adrenalectomy but, at least in rats, not for increasing the number of corticotrophs. The lack of colocalization of ACTH in mitotic cells suggests that recruitment of corticotrophs during adrenalectomy occurs from undifferentiated cells.

Environmental lead exposure during early life alters granule cell neurogenesis and morphology in the hippocampus of young adult rats

Exposure to environmentally relevant levels of lead (Pb 2+) during early life produces deficits in hippocampal synaptic plasticity in the form of long-term potentiation (LTP) and spatial learning in young adult rats [Nihei MK, Desmond NL, McGlothan JL, Kuhlmann AC, Guilarte TR (2000) N-methyl-D-aspartate receptor subunit changes are associated with lead-induced deficits of long-term potentiation and spatial learning. Neuroscience 99:233–242; Guilarte TR, Toscano CD, McGlothan JL, Weaver SA (2003) Environmental enrichment reverses cognitive and molecular deficits induced by developmental lead exposure. Ann Neurol 53:50–56]. Other evidence suggests that the performance of rats in the Morris water maze spatial learning tasks is associated with the level of granule cell neurogenesis in the dentate gyrus (DG) [ Drapeau E, Mayo W, Aurousseau C, Le Moal M, Piazza P-V, Abrous DN (2003) Spatial memory performance of aged rats in the water maze predicts level of hippocampal neurogenesis. Proc Natl Acad Sci U S A 100:14385–14390]. In this study, we examined whether continuous exposure to environmentally relevant levels of Pb 2+ during early life altered granule cell neurogenesis and morphology in the rat hippocampus. Control and Pb 2+-exposed rats received bromodeoxyuridine (BrdU) injections (100 mg/kg; i.p.) for five consecutive days starting at postnatal day 45 and were killed either 1 day or 4 weeks after the last injection. The total number of newborn cells in the DG of Pb 2+-exposed rats was significantly decreased (13%; P<0.001) 1 day after BrdU injections relative to controls. Further, the survival of newborn cells in Pb 2+-exposed rats was significantly decreased by 22.7% ( P<0.001) relative to control animals. Co-localization of BrdU with neuronal or astrocytic markers did not reveal a significant effect of Pb 2+ exposure on cellular fate. In Pb 2+-exposed rats, immature granule cells immunolabeled with doublecortin (DCX) displayed aberrant dendritic morphology. That is, the overall length-density of the DCX-positive apical dendrites in the outer portion of the DG molecular layer was significantly reduced up to 36% in the suprapyramidal blade only. We also found that the area of Timm’s-positive staining representative of the mossy fibers terminal fields in the CA3 stratum oriens (SO) was reduced by 26% in Pb 2+-exposed rats. These findings demonstrate that exposure to environmentally relevant levels of Pb 2+ during early life alters granule cell neurogenesis and morphology in the rat hippocampus. They provide a cellular and morphological basis for the deficits in synaptic plasticity and spatial learning documented in Pb 2+-exposed animals.

Pten and the Brain: Sizing up Social Interaction

In a comprehensive set of experiments in this issue of Neuron, Kwon et al. demonstrate that conditional inactivation of Pten leads to behavioral abnormalities and neuropathological changes. Pten mutants displayed reduced social interaction and heightened responses to sensory stimuli. Additionally, neuronal hypertrophy, as well as macrocephaly was observed. Based on rare human mutations in PTEN and the PI3K pathway, the authors suggest they have produced a potential animal model of autism with macrocephaly.

Enhanced neurogenesis during trimethyltin-induced neurodegeneration in the hippocampus of the adult rat

The occurrence of neurogenesis in the hippocampus of the adult rat during trimethyltin (TMT)-induced neurodegeneration was investigated using bromodeoxyuridine (BrdU). Fifteen days after TMT intoxication, BrdU-labeled cells were significantly more numerous in the hippocampus of treated animals, gradually decreasing towards the control value 21 days after intoxication in the dentate gyrus (DG), while in the CA3/hilus region BrdU-labeled cells were still more numerous in TMT-treated rats. In order to investigate the fate of newly-generated cells double labeling experiments using neuronal or glial markers were performed. Colocalization of the neuronal marker NeuN was detected in many BrdU-positive cells in the DG, while in the CA3/hilus region no colocalization of NeuN and BrdU could be observed. No colocalization of BrdU and the astroglial marker GFAP or the microglial marker OX-42 was detected either in the DG and or in the CA3/hilus region. The results indicate an enhancement of endogenous neurogenesis in the hippocampus during TMT-induced neurodegeneration, with the development of a subpopulation of regenerated cells into neurons in the DG, while in the CA3/hilus region the population of newly-generated cells should be regarded as undifferentiated.

Neurogenesis in hippocampal slice cultures

A major challenge in studying neurogenesis in the adult brain is gaining access to neural stem cells for experimental manipulation. We developed an approach utilizing mouse hippocampal organotypic cultures to characterize neurogenesis under controlled conditions. After 2 weeks in culture, double immunostaining using the mitotic marker BrdU and cell type-specific markers revealed persistent proliferation of various cell types. The birth of new neurons was restricted to a third subgranular germinal zone as shown by analysis of the expression pattern of the proneural transcription factor neurogenin-2 and colocalization of BrdU with neuronal phenotypic markers. The regional distribution of newly born neurons closely resembled that observed in vivo in the adult hippocampus. Furthermore, neurogenesis was increased by chronic application of epidermal growth factor (EGF) and abolished by adding serum to the culture medium. Our study therefore establishes the hippocampal slice culture as a promising ex vivo model for investigating neurogenesis.