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The Effect of Advanced Motherhood on Newborn Offspring’s Hippocampal Neural Stem Cell Proliferation

¹Department of Physiology, Zhengzhou University, Zhengzhou, Henan 450001, China
²Stem Cell Research Center of Medical College, Zhengzhou University, Zhengzhou, Henan 450052, China
³Department of Pathophysiology, Zhengzhou University, Zhengzhou, Henan 450001, China

Received 10 May 2016; Revised 9 July 2016; Accepted 12 July 2016

Academic Editor: Oliver von Bohlen und Halbach

Copyright © 2016 Bo Li et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Objective. To investigate the effect of advanced motherhood on rat hippocampal neural stem cell proliferation. Methods. Female parents were subdivided into control and old mother group by age, and neural stem cells were cultured from hippocampal tissues for 24 h newborn offspring. The diameter and numbers of neurospheres were examined by microscopy, and differences in proliferation were examined by EdU immunofluorescence, CCK-8 assay, and cell cycle analysis. Results. The number of neurospheres in the old mother group after culture was lower than the control group. Additionally, neurospheres’ diameter was smaller than that of the control group (). The EdU positive rate of the old mother group was lower than that of the control group (). CCK-8 assay results showed that the absorbance values for the old mother group were lower than that of the control group at 48 h and 72 h (). The proportions of cells in the S and G2/M phases of the cell cycle for the older mother group were less than that found for the control group (). Conclusion. The proliferation rates of hippocampal NSCs seen in the older mother group were lower than that seen in the control group.

1. Introduction

Delayed child-bearing has become increasingly common in human reproduction due to increasing trends in economic and social pressures in today’s world. Moreover, the delayed child-bearing age might affect mental development of the offspring—this possibility has attracted increasing attention recently [1]. An age greater than 30 at the time of motherhood is known to affect the quality of the offspring [2], increase the incidence of hypospadias [3], increasing the likelihood of autism spectrum disorders [4, 5], and affecting the intellectual disability [6].

In mouse models, studies have found that offspring of older mothers showed a higher mortality rate before the weaning period; in addition, not only was low birth weight seen, but also spontaneous activity and learning deficits are seen during adolescence [7]. However, other studies have shown quite the opposite effects and have demonstrated that the mother’s age at pregnancy had no effect on intelligence evaluation of the newborn baby [8, 9]. Moreover, even in children of older pregnant mothers, higher intelligence test scores (IQ) were seen [10]. Therefore, whether child intelligence if adversely affected by onset of motherhood age remains in dispute.

The ability of learning and memory can reflect differences in intelligence, which is associated with new neuronal generation in the hippocampus [11, 12]. Delayed motherhood was found to lead to unique patterns of rat hippocampal gene expression in offspring as compared to the reference group [13]. Neural stem cells are highly localized to the hippocampal dentate gyrus. Studies showed that, with increased neural stem cell division and proliferation, the stronger the ability to learn and develop effective memory [14, 15]. To date, the effect of delayed motherhood on hippocampal neural stem cell proliferation has not been previously reported.

By determining proliferative characteristics of the offspring between younger mothers and older mothers, the aim of this study was to examine differences at the cellular level.

2. Materials and Methods

2.1. Materials

2.1.1. Animals

Newborn 24-hour-old Sprague-Dawley male rats (Animal Center of Zhengzhou University, Zhengzhou, China) were used in these experiments. Virgin females were subdivided into two groups by age: (1) 3 months old (the control group, ) and (2) 12 months old (the old mother group, ); females in the two groups were housed with randomly selected 3-month-old male rats.

All rat experiments were conducted in terms of the “Guidance on the Care and Use of Laboratory Animals” (2006, People’s Republic of China National Science and Technology Committee Notice). Animal studies were approved by the Animal Ethics Committee of Zhengzhou University.

2.1.2. Reagents

The reagents used in this study were 0.25% Trypsin-EDTA (1x); DMEM culture medium; fetal bovine serum (FBS); trypsin-EDTA and B27 (Gibco, USA); basic fibroblast growth factor (bFGF); epidermal growth factor (EGF) (PeproTech, London, UK); phycoerythrin- (PE-) conjugated anti-nestin antibody, which is a neural progenitor specific marker; propidium iodide (PI)/RNase staining buffer; Cytofix/Cytoperm kit (BD biosciences, Franklin Lakes, NJ, USA); 4,6-diamidino-2-phenylindole (DAPI) Nuclear Labeling Kit (GenView, Houston, TX, USA); cell proliferation assay (CCK-8 kit; KeyGEN BioTECH Corp., Ltd. Shanghai, China); and 5-ethynyl-2′-deoxyuridine (EdU) in vitro DNA proliferation assay kit (Rui Bo Guangzhou Biotechnology Limited Company, China).

2.2. Isolation, Purification, and Culture of NSCs

Neurospheres were generated from isolated NPCs in the hippocampal area of postnatal 24-hour-old rat. Briefly, rat brains were coronally sectioned, and the hippocampal area was obtained, which was followed by tissue and cellular dissociation. Isolated cells were cultured at a density of 5 × 10⁵ cells/mL in DMEM-F12 proliferation medium containing 2% B27 supplement, 20 ng/mL bFGF, and 20 ng/mL EGF. The cultures were observed and photographed daily under a phase contrast microscope (Model CKX41, Olympus, Japan). After 2 passages, cells were cultured for 72 and 96 h, Olympus imaging analysis system was employed to observe of neurospheres, and the number and diameter of neurospheres were counted and measured separately in 10 randomly selected microscopic fields (100x) for each flask.

2.3. Immunocytochemistry

Neurospheres were seeded onto coverslips that were precoated with poly-L-lysine (0.01 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) for 5 h until the neurospheres had adhered, following which they were washed in PBS three times and fixed in 4% paraformaldehyde (PFA) for 30 min. Fixed cells were blocked in 0.3% Triton X-100 supplemented with PBS and 5% goat serum for 1 h at room temperature. Slides were washed in PBS three times and incubated with PE-conjugated nestin antibody (1 : 100) for 2 h at 37°C in the dark. Nuclei were counterstained with DAPI (1 : 10000) for 10 min at room temperature. Epifluorescence observation and photodocumentation were done using an Olympus BX51 microscope (Olympus, Japan) that was equipped with a Spot™ digital camera (Diagnostic Instrument Inc., USA).

2.4. Flow Cytometry

Neurospheres were dissociated into single cells and resuspended in PBS. After centrifugation (179 ×g, 5 min, room temperature), cells were treated in accordance with the instructions that accompanied the BD Cytofix/Cytoperm assay kit. In this assay cells were incubated with 500 μL of fixation/permeabilization solution that were then incubated for 20 min in the dark at room temperature. After centrifugation, cell pellets were incubated with 2 mL of BD perm/wash buffer for 10 min and then incubated with PE-conjugated-nestin antibodies (1 : 20) for 1 h at 4°C in the dark. Cells were then washed and resuspended in 300 μL of PBS containing 1% paraformaldehyde. The ratio of nestin positive cells were analyzed by flow cytometry (BD FACSCanto II) with Data-Interpolating Vibrational Analysis (DIVA) software v. 5.0.

2.5. EdU Staining

Neurospheres were dissociated into single cells by Accutase and resuspended in PBS. The cells were seeded at a density of 1 × 10⁵ cells/mL into 24-well culture plates. EdU staining was conducted using a Cell-Light™ EdU staining assay kit, according to the manufacturer’s protocol. Cells were incubated with EdU (1 : 5000) for 24 h and then resuspended with DMEM-F12 proliferation medium and seeded onto coverslips that were precoated with poly-L-lysine and then incubated for 6 h in 37°C, 5% CO₂. The culture supernatant was then removed, and 2 mg/mL glycine solution was added for 10 min at room temperature. Next, cells were rinsed in PBS for 5 min and the sections were permeabilized with 0.5% Triton X-100 in PBS for 10 min and washed twice with PBS for 10 min per wash. Cells were incubated with Apollo staining reaction solution for 30 min in the dark. Cells were washed twice in PBS that contained 0.5% Triton X-100 for 10 min per wash. Next, cells were counterstained with Hoechst 33342 for 30 min in the dark to stain the nuclei. The slides were then washed twice with PBS for 3 min per wash and observed immediately by fluorescence microscopy at a magnification of ×400.

2.6. CCK-8 Assay

Neurospheres were dissociated into single cells by Accutase and resuspended in PBS. The cells were seeded at a density of 1 × 10⁵ cells/mL in nine culture plates. The growth rates of the cells were then determined by CCK-8 assay. Next, cells were incubated in 10 μL CCK-8 working solution at 0, 24, 48, and 72 h, followed by incubation for 2 h at 37°C. The absorbance was finally measured at 450 nm using a model 3550 microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

2.7. Cell Cycle Analysis by Flow Cytometry

Neurospheres were dissociated into single cells by Accutase and resuspended in PBS. Cells were then washed three times in PBS and then resuspended in 70% chilled ethanol and fixed at −20°C overnight. The cells were then washed and resuspended in 500 L PI/RNase Staining Buffer and incubated at room temperature for 30 min. Cell cycle analysis was performed by flow cytometry. Data from 30,000 cells were collected for each data file (Figure 4).

2.8. Statistical Analysis

Data are described as mean ± standard deviation. Statistical analysis of the data was carried out by Student’s t-test using the SPSS v. 13.0 software program (SPSS, Inc., Chicago, IL, USA). An alpha value of was considered a statistically significant difference.

3. Results

3.1. NSC Culture and Detection

Neurospheres could be seen after hippocampal neural stem cell primary culture for three days, and the third generation of the cultured cells were observed. The number of neurospheres in the old mother group at 72 h and 96 h were lower than that seen in the control group, that is, versus and versus , respectively (Figure 1(a)). The diameters of the neurospheres were also recorded in the old mother group and were mm at 72 h and at 96 h as compared to that seen in the control group, which were mm and , respectively () (Figure 1(b)).

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