Mesenchymal stem cells promote proliferation of endogenous neural stem cells and survival of newborn cells in a rat stroke model

Mesenchymal stem cells (MSCs) secrete bioactive factors that exert diverse responses in vivo. In the present study, we explored mechanism how MSCs may lead to higher functional recovery in the animal stroke model. Bone marrow-derived MSCs were transplanted into the brain parenchyma 3 days after induction of stroke by occluding middle cerebral artery for 2 h. Stoke induced proliferation of resident neural stem cells in subventricular zone. However, most of new born cells underwent cell death and had a limited impact on functional recovery after stroke. Transplantation of MSCs enhanced proliferation of endogenous neural stem cells while suppressing the cell death of newly generated cells. Thereby, newborn cells migrated toward ischemic territory and differentiated in ischemic boundaries into doublecortin+ neuroblasts at higher rates in animals with MSCs compared to control group. The present study indicates that therapeutic effects of MSCs are at least partly ascribed to dual functions of MSCs by enhancing endogenous neurogenesis and protecting newborn cells from deleterious environment. The results reinforce the prospects of clinical application using MSCs in the treatment of neurological disorders.     

Ischemic preconditioning in the rat hippocampus increases antioxidant activities but does not affect the level of hydroxyl radicals during subsequent severe ischemia

Several studies have demonstrated that ischemic preconditioning increases superoxide dismutase activity, but it is unclear how ischemic preconditioning affects events downstream of hydrogen peroxide production during subsequent severe ischemia and reperfusion in the hippocampus. To answer this question, we investigated whether ischemic preconditioning in the hippocampal CA1 region increases the activities of antioxidant enzymes glutathione peroxidase and catalase, resulting in a decrease in the level of hydroxyl radicals during subsequent severe ischemia-reperfusion. Transient forebrain ischemia was induced by four-vessel occlusion in rats. Ischemic preconditioning for 3 min or a sham operation was performed and a 15-min severe ischemia was induced three days later. Ischemic preconditioning preserved the CA1 hippocampal neurons following severe ischemia. The concentration of 2,3-dihydroxybenzoic acid, an indicator of hydroxyl radical, was measured using in vivo microdialysis technique combined with HPLC. The ischemia-induced increase in the ratio of 2,3-dihydroxybenzoic acid concentration relative to baseline did not differ significantly between preconditioned and control groups. On the other hand, activities of the antioxidant enzymes glutathione peroxidase-1 and catalase were significantly increased at 3 days after ischemic preconditioning in the hippocampus. Our results suggest that, in preconditioned rats, while hydrogen peroxide is generated from severe ischemia, the activity of catalase and glutathione peroxidase-1 is correspondingly increased to eliminate the excessive hydrogen peroxide. However, our results show that the enhanced activity of these antioxidant enzymes in preconditioned rats is not sufficient to decrease hydroxyl radical levels during subsequent severe ischemia-reperfusion.     

Singlet oxygen, but not oxidizing radicals, induces apoptosis in HL-60 cells

Oxidizing species (OS), produced by photosensitization or derived from cytotoxic agents, activate apoptotic pathways. We investigated whether two different OS, formed at the same subcellular sites, have equivalent ability to initiate apoptosis in HL-60 cells. Our previous work showed that absorption of visible light by rose bengal (RB) produces singlet oxygen exclusively, whereas absorption of ultraviolet A produces RB-derived radicals in addition to singlet oxygen. Singlet oxygen, but not the RB-derived radicals, induced nuclear condensation and DNA fragmentation into nucleosome-size fragments in a dose dependent manner. In contrast, the RB-derived radicals caused greater lipid oxidation than singlet oxygen. These results indicate that different OS, produced at the same subcellular sites, do not have the same ability to induce apoptosis and that the ability of an OS to initiate lipid oxidation does not necessarily correlate with its ability to induce apoptosis.     

Gardenia fruit extract does not stimulate the proliferation of cultured vascular smooth muscle cells, A10

We investigated the effect of a hot water extract from Gardenia fruit (Gardenia jasminoides Ellis) (GFE), which has a stimulatory effect on endothelial cell proliferation, on the proliferation of A10 cells, an established cell line of vascular smooth muscle cell from murine aorta in a culture system. GFE did not change the number of A10 cells after a 48 h culture. GFE significantly increased the incorporation of [3H]thymidine and [14C]leucine into the acid-soluble fraction of bovine aortic endothelial cell layers, but significantly decreased that of A10 cells. These results suggested that GFE stimulates the proliferation of endothelial cells but not of A10 cells. In the endothelial cell culture, GFE significantly increased the accumulation of basic fibroblast growth factor, which is an autocrine for endothelial cell proliferation in medium and low-affinity (glycosaminoglycans-binding) fractions, while A10 cells did not produce a significant amount of the factor. Since it is postulated that a selective stimulation of endothelial cell proliferation by increasing the production of basic fibroblast growth factor is appropriate for prevention of arteriosclerosis and thrombosis, GFE may contain a beneficial component as a useful drug.     

Mechanical stretch promotes proliferation of rat bone marrow mesenchymal stem cells

Tissues and cells in the body are continuously exposed to a complex mechanical environment. Mechanical stimulations are critical to morphological, developmental and functional states of living cells, and the fashion of the mechanical stimulation applied to the cells is supposed to be extremely important for the induced cell response and function. In this study, we investigated whether mechanical stretch regulates and promotes proliferation of rat bone marrow mesenchymal stem cells (rMSCs) in vitro. rMSCs from rat bone marrow were isolated, purified and subjected to a cyclic equiaxial stretch treatment, and then MTT assay was adopted and expression of c-fos gene was measured by RT-PCR to access cell proliferation. The results demonstrated that OD values of rMSCs increased in a time-dependent and magnitude-dependent manner after exposure to 1 Hz stretch within 15–60 min and 2–8% strain. Expression of c-fos gene in rMSCs subjected to stretch treatment (1 Hz, 8% strain and 60 min) is significantly higher than that of unstimulated control cells. These results suggest that mechanical stretch plays an important role in regulating the cell growth and proliferation, and an appropriate mechanical stretch treatment could promote proliferating capacity of rMSCs.     

Paecilomyces variotii extract increases lifespan and protects against oxidative stress in Caenorhabditis elegans through SKN-1, but not DAF-16

A natural extract from Paecilomyces variotii (P. variotii extract, PVE), an endophytic fungus, has been used widely to improve agricultural crop performance and control multiple plant pathogens. Most recent studies focused on its application as a plant growth promoter, while relatively few studies have been reported on the antioxidant potential in vivo and the underlying mechanism. The present study was designed to determine the antioxidant activities of PVE and its mechanisms using Caenorhabditis elegans. Results showed that, compared to the solvent control, PVE at 1.0, 10 and 100 ng/mL significantly extended the lifespan of C. elegans by 36.60%, 59.80% and 53.30%, respectively. PVE at 10 ng/mL consistently promoted nematodes growth, but all treatments did not influence nematode fecundity, locomotion behavior, and pharyngeal pumping. Furthermore, PVE at the three tested concentrations significantly reduced accumulation of reactive oxygen species (ROS), lipofuscin, lipid and malondialdehyde (MDA) content, meanwhile significantly promoted activities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) in the nematodes. Compared with the solvent control, PVE up-regulated gene expression of skn-1, mev-1, sod-3, and daf-2, but significantly down-regulated the expression of nhr-49 and daf-16. Further evidence revealed that PVE at the three concentrations significantly promoted nuclear localization of SKN-1, but not affected that of DAF-16, indicating the complex roles of DAF-16 and SKN-1 in stress resistance and longevity regulation. Overall, our results demonstrated that SKN-1 played a critical role in increasing lifespan of C. elegans and protecting the nematodes from oxidative stress, independent of DAF-16.     

Ultraviolet B but not A radiation activates suppressor B cells in draining lymph nodes

Immunosuppressive doses of solar-simulated UV radiation activate lymph node B cells that can suppress primary immunity by inhibiting the function of dendritic cells. The aim of this study was to determine the waveband responsible for activation of these suppressor B cells. We exposed C57BL/6 mice to various doses of either UVA or UVB radiation and analyzed the number and activation state of lymph node antigen-presenting cells (APC). Immunosuppressive doses of UVB but not UVA activated B cells as assessed by major histocompatibility complex II (MHC II) expression and doubled their numbers in draining lymph nodes. Higher doses of UVA that were not immunosuppressive actually suppressed B cell activation. Our results show that UVA and UVB suppress systemic immunity via different mechanisms. Lymph node B cells are activated in response to immunosuppressive doses of UVB but not UVA. Thus, the activation state of lymph node APC appears to be important for UV immunomodulation.     

5-aminolevulinic acid, but not 5-aminolevulinic acid esters, is transported into adenocarcinoma cells by system BETA transporters

5-aminolevulinic acid (5-ALA) and its ester derivatives are used in photodynamic therapy as precursors for the formation of photosensitizers. This study relates to the mechanisms by which 5-ALA is transported into cells. The transport of 5-ALA has been studied in a human adenocarcinoma cell line (WiDr) by means of [14C]-labeled 5-ALA. The rate of uptake was saturable following Michaelis-Menten kinetics (K(m) = 8-10 mM and Vmax = 18-20 nmol.(mg protein x h)-1), and Arrhenius plot of the temperature-dependent uptake of 5-ALA was characterized by a single discontinuity at 32 degrees C. The activation energy was 112 kJ.mol-1 in the temperature range 15 degrees-32 degrees C and 26 kJ.mol-1 above 32 degrees C. Transport of 5-ALA was Na+ and partly Cl(-)-dependent. Stoichiometric analysis revealed a Na+:5-ALA coupling ratio of 3:1. With the exception of valine, methionine and threonine, zwitterionic and basic amino acids inhibited the transport of 5-ALA. 5-ALA methyl ester was not an inhibitor of 5-ALA uptake. The transport was most efficiently inhibited, i.e. by 65-75%, by the beta-amino acids, beta-alanine and taurine and by gamma-aminobutyric acid (GABA). Accordingly, 5-ALA, but not 5-ALA methyl ester, was found to inhibit cellular uptake of [3H]-GABA and [14C]-beta-alanine. Protoporphyrin IX (PpIX) accumulation in the presence of 5-ALA (0.3 mM) was attenuated 85% in the presence of 10 mM beta-alanine, while PpIX formation in cells treated with 5-ALA methyl ester (0.3 mM) or 5-ALA hexyl ester (4 microM) was not significantly influenced by beta-alanine. Thus, 5-ALA, but not 5-ALA esters, is transported by beta-amino acid and GABA carriers in this cell line.     

Chronic activation of CREB and p90RSK in human epileptic hippocampus

Mesial temporal lobe epilepsy (MTLE) is associated with severe neuronal death and reactive gliosis in hippocampus. However, the molecular mechanisms underlying these pathological changes remain unanswered. ERK has been reported chronically activated in reactive glia of human epileptic hippocampus. In the present study, we investigated which of the downstream signaling molecules of ERK would be involved in MTLE. Western blot analysis demonstrated that CREB and p90RSK were strongly activated in MTLE patients. Increase in the active forms of CREB and p90RSK resulted not only from the increase in their phosphorylation levels but also from the increase in the protein levels. Activation of CREB and p90RSK was noted in the whole subfields of hippocampus with Ammon's horn sclerosis (AHS) representing a distinctive cellular distribution. However, the common major change was present in proliferating reactive astrocytes. In contrast, their activation was not significant in adjacent temporal lobes despite the presence of a number of astrocytes expressing high levels of GFAP. Our results demonstrate that chronic activation CREB and p90RSK in the epileptic hippocampus may be closely associated with the histopathological changes of AHS.     

Growth factor-expressing human neural progenitor cell grafts protect motor neurons but do not ameliorate motor performance and survival in ALS mice

Neural progenitor cells (NPs) have shown several promising benefits for the treatment of neurological disorders. To evaluate the therapeutic potential of human neural progenitor cells (hNPs) in amyotrophic lateral sclerosis (ALS), we transplanted hNPs or growth factor (GF)-expressing hNPs into the central nervous system (CNS) of mutant Cu/Zn superoxide dismutase (SOD1^G93A) transgenic mice. The hNPs were engineered to express brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), VEGF, neurotrophin-3 (NT-3), or glial cell-derived neurotrophic factor (GDNF), respectively, by adenoviral vector and GDNF by lentiviral vector before transplantation. Donor-derived cells engrafted and migrated into the spinal cord or brain of ALS mice and differentiated into neurons, oligodendrocytes, or glutamate transporter-1 (GLT1)-expressing astrocytes while some cells retained immature markers. Transplantation of GDNF- or IGF-1-expressing hNPs attenuated the loss of motor neurons and induced trophic changes in motor neurons of the spinal cord. However, improvement in motor performance and extension of lifespan were not observed in all hNP transplantation groups compared to vehicle-injected controls. Moreover, the lifespan of GDNF-expressing hNP recipient mice by lentiviral vector was shortened compared to controls, which was largely due to the decreased survival times of female animals. These results imply that although implanted hNPs differentiate into GLT1-expressing astrocytes and secrete GFs, which maintain dying motor neurons, inadequate trophic support could be harmful and there is sexual dimorphism in response to GDNF delivery in ALS mice. Therefore, additional therapeutic approaches may be required for full functional recovery.     

Kale extract increases glutathione levels in V79 cells, but does not protect them against acute toxicity induced by hydrogen peroxide

This study aims to evaluate the antioxidant potential of extracts of Brassica oleracea L. var. acephala DC. (kale) and several materials of Pieris brassicae L., a common pest of Brassica cultures using a cellular model with hamster lung fibroblast (V79 cells) under quiescent conditions and subjected to H?O? induced oxidative stress. Cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and glutathione was determined by the 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)-oxidized glutathione (GSSG) reductase recycling assay. The phenolic composition of the extracts was also established by HPLC-DAD. They presented acylated and non acylated flavonoid glycosides, some of them sulfated, and hydroxycinnamic acyl gentiobiosides. All extracts were cytotoxic by themselves at high concentrations and failed to protect V79 cells against H?O? acute toxicity. No relationship between phenolic composition and cytotoxicity of the extracts was found. Rather, a significant increase in glutathione was observed in cells exposed to kale extract, which contained the highest amount and variety of flavonoids. It can be concluded that although flavonoids-rich extracts have the ability to increase cellular antioxidant defenses, the use of extracts of kale and P. brassicae materials by pharmaceutical or food industries, may constitute an insult to health, especially to debilitated individuals, if high doses are consumed.     

Changes in neurite outgrowth but not in cell division induced by low EMF exposure: influence of field strength and culture conditions on responses in rat PC12 pheochromocytoma cells

The effects of low electromagnetic field (EMF) exposure (4.5-15.8 microT, 50 Hz AC) on neurite outgrowth and cell division in rat PC12 pheochromocytoma cells were examined. The study involved two separate experimental series in which culture conditions during exposure to the magnetic fields differed. In series 1 (14 experiments in which culture conditions were not strongly conducive to cell differentiation [15% serum]), exposure to 4.5-8.25 microT for 23 h significantly inhibited neurite outgrowth by 21.5 +/- 1.3% (by Manova, p = 0.003). In contrast, in series 2 (12 experiments in which culture conditions promoted cellular differentiation [4% serum]), exposure to 4.35-8.25 microT for 23 h significantly stimulated neurite outgrowth by 16.9 +/- 1.1% (by Manova, p = 0.009). Thus, in both series, exposure to a narrow range of low EMF has significant, but opposite effects on neurite outgrowth. Exposure to higher fields, 8.25-12.5 microT (series 1) and 8.25-15.8 microT (series 2) had no significant effect on neurite outgrowth. These data, when considered with other reports, suggest that neuronal differentiation can be altered by low level EMF exposure. While this may not be detrimental, it merits further research. At present, the reasons for the significant changes in neurite outgrowth being confined to the same narrow field strength are unclear. As stated above, culture conditions in series 2 were more conducive to cell differentiation than those in series 1. This is reflected in the lower number of cells in control samples in series 2, at the end of the 23-h incubation, than in series 1 (- 16.9 +/- 1.7%, p = 0.003). As the same numbers were plated in both series, the medium used in series 1 allows more of the PC12 cells to divide; this is consistent with some cells reverting to a non-neuronal adrenal chromaffin phenotype [L. Greene, A. Tischler. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. U. S. A., 73 (1976) 2424-2426]. Exposure to both ranges of magnetic fields (4.35-8.25 and 8.25-15.8 microT) has no effect on cell division. Thus, there is no evidence in this study that there is a mitogenic effect arising from low EMF exposure.     

Sustained release of acetylcholine in rat hippocampus using a polyanhydride drug-delivery system

A biodegradable polymer drug-delivery system has been developed for the selective localized application of agents to brain parenchyma. The copolymer of poly[bis(p-carboxyphenoxy)propane] anhydride and sebacic acid (PCPP–SA) was impregnated with [³H]-acetylcholine (ACh) to form 1–5 μm microspheres. Drug-loaded microspheres were implanted into hippocampus bilaterally in 25 rats, and brain sections processed for autoradiography in groups of five animals at 2, 5, 10, 20 and 40 days, respectively. By densitometric analysis, the concentration of radiolabelled ACh in polymer and adjacent hippocampus rapidly decreased between 2 and 5 days, after which a gradual decrease in [³H]-ACh was observed up to 40 days. Between 2 and 40 days the concentration of radiolabelled ACh was reduced by 25.8% in polymer matrix and 40.1% in hippocampus. The spread of [³H]-label into adjacent brain parenchyma showed a similar temporal relationship, with initially wider dispersion at 2 days (44±3 μm), then a linear decrease in dispersion over the remaining period (10±0.9 μxm at 40 days), suggesting bulk flow of the radiolabel into hippocampus. Brain parenchyma showed only a minimal inflammatory reaction to the polyanhydride implants over all time periods. Polyanhydrides can provide localized continuous release of ACh to brain parenchyma, and may potentially be used to deliver various agents to brain in a number of clinical and experimental applications.     

保存条件对大鼠海马组织总RNA质量的影响

从大鼠海马组织提取总RNA后,测定了不同时间和温度下保存及3次循环冻融后RNA的浓度和纯度,并分析了其完整性;探讨了保存时间、保存温度及反复冻融对大鼠海马组织总RNA质量的影响.结果表明,不同条件下保存及冻融3次后RNA浓度较首次检测值均有所降低,4℃下保存60d标本中的RNA浓度明显低于对照组(P<0.05),其他组未见显著性差异;各组RNA的OD 260/OD280在1.8～2.0之间.在-20℃和-70℃下分别保存30d和60d,以及24h冻融3次后的样品RNA的完整性较好;但4℃下保存的两组样品的RNA发生分解.RNA在4℃和-20℃下分别保存30d和60d后,相应的RE[(不同条件下保存后的样品浓度-初始浓度)/初始浓度]值均大于15%,而-70℃下保存30d、60d以及在24h内冻融3次后的RE值均小于15%.据此可知,大鼠海马组织总RNA样品可在-70℃下保存60d,反复冻融3次后总RNA质量基本不变.     

Induction of Bis,a Bcl-2-binding protein,in reactive astrocytes of the rat hippocampus following kainic acid-induced seizure

The expression of Bis (also called Bag-3), a Bcl-2-binding protein, was investigated in the rat kainic acid (KA) model of temporal lobe epilepsy. Western blot analysis showed a significant increase in the expression levels of Bis protein in the hippocampus following the systemic administration of KA. Bis immunoreactivity increased preferentially in the CA1 and CA3 regions, as well as in the hilar region of the dentate gyrus. Experiments with double immunofluorescence revealed that, in KA-administered rats, the cells expressing Bis were GFAP-expressing reactive astrocytes. The increase in Bis immunoreactivity was accompanied by increased Bcl-2 in reactive astrocytes in the striatum radiatum, whereas Bcl-2 immunoreactivity in pyramidal neurons was not affected. These results of the co-expression of Bis and Bcl-2 in reactive astrocytes in this seizure model suggest that Bis might modulate the glial reaction under excitotoxic brain injury, probably by interacting with Bcl-2.     

A simple elastic membrane-based microfluidic chip for the proliferation and differentiation of mesenchymal stem cells under tensile stress

This work presents a simple membrane-based microfluidic chip for the investigation of proliferation and differentiation of mesenchymal stem cells (MSCs) under mechanical stimuli. The cyclic tensile stress was generated by the deformation of elastic PDMS membrane sandwiched between the two layer microfluidic chip via actuated negative pressure, and the cultured MSCs on membrane were subjected to different orders of tensile stress. The results suggest that mechanical stimuli are attributed to the different phenomena of MSCs in cell proliferation and differentiation. The higher tensile stress (>3.5) promoted obvious proliferation, osteogenesis and reduced adipogenesis in MSCs, indicating the possible regulative role of tensile stress in modifying the osteogenesis/adipogenesis balance in the development of tissue organ.     

Effect of fatty acid methyl esters from plastrum testudinis on proliferation of rat bone mesenchymal stem cells

The ointment of plastrum testudinis was extracted using petroleum ether, ether and dichloromethane sequentially and the extracts were methyl-esterified. The effects on the proliferation of bone marrow mesenchymal stem cells (bMMSCs) were examined by MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay and flow cytometry analysis. The volatile components of the samples were studied by gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC). The results show that the methyl-esterified parts can promote the proliferation of stem cells and they all contain palmitic acid methyl ester. Palmitic acid methyl ester can promote proliferation when the concentration was 0.15 μg/μL. It may be concluded that the palmitic acid methyl ester is important for the methyl-esterified parts that have effects on proliferation. __________ Translated from Chinese Journal of Analytical Chemistry, 2007, 35 (10): 1400–1404 [译自: 分析化学]