Microchip encapsulation and microRNA-7 overexpression of trabecular meshwork mesenchymal stem/stromal cells improve motor function after spinal cord injury.

Manipulation of stem cells and microencapsulation through microfluidic chips has shown more promising results in treating complex conditions, such as spinal cord injury (SCI), than traditional treatments. This study aimed to investigate the potency of neural differentiation and its therapeutic role in SCI animal model of trabecular meshwork mesenchymal stem/stromal cells (TMMSCs) via miR-7 overexpression and microchip-encapsulated. TMMSCs are transduced with miR-7 via a lentiviral vector (TMMSCs-miR-7[+]) and encapsulated in alginate-reduced graphene oxide (alginate-rGO) hydrogel via a microfluidic chip. Neuronal differentiation of transduced cells in hydrogel (3D) and tissue cultures plate (2D) was assessed by expressing specific mRNAs and proteins. Further evaluation is being carried out through 3D and 2D TMMSCs-miR-7(+ and -) transplantation into the rat contusion SCI model. TMMSCs-miR-7(+) encapsulated in the microfluidic chip (miR-7-3D) increased nestin, ß-tubulin III, and MAP-2 expression compared with 2D culture. Moreover, miR-7-3D could improve locomotor behavior in contusion SCI rats, decrease cavity size, and increase myelination. Our results revealed that miR-7 and alginate-rGO hydrogel were involved in the neuronal differentiation of TMMSCs in a time-dependent manner. In addition, the microfluidic-encapsulated miR-7 overexpression TMMSCs represented a better survival and integration of the transplanted cells and the repair of SCI. Collectively, the combination of miR-7 overexpression and encapsulation of TMMSCs in hydrogels may represent a promising new treatment for SCI.

Effect of selegiline as a monomine oxidase B inhibitor on the expression of neurotrophin mRNA levels in a contusion rat model of spinal cord injury.

OBJECTIVE: Spinal cord injury (SCI) is followed by a cascade of events at the site of injury, including vascular ischemia, an increase in free radicals, inflammation, and neuronal death. In these individuals, protection of nerves and supporting cells, as well as prevention of neuronal damage, may improve recovery opportunities. Neurotrophins are a family of polypeptides that regulate nerve differentiation, growth, and survival. Selegiline is a selective monoamine oxidase B (MAO-B) inhibitor used to treat Parkinson's disease. Selegiline has been found to have neuroprotective properties and may be useful for the expression of neurotrophins. The aim of this study was to evaluate the expression levels of neurotrophin genes in spinal cord rats treated with selegiline. METHODS: Rats were divided into four groups: injury (control), laminectomy, sham (injured rat received 1 ml saline intraperitoneally) and treatment (injured rat received 5 mg/kg selegiline intraperitoneally for 7 days; once a day). The BBB scale (Basso, Beattie and Bresnahan) was performed once a week for 4 weeks to assess motor function after a spinal cord injury. On day 28 after SCI, the rat was sacrificed and the spinal cord lesion removed. A real-time PCR approach was used to assess neurotrophin gene expression. RESULTS: The results showed that administration of selegiline improves locomotor function and increases mRNA levels of BDNF, GDNF, NT-3, and NT-4. CONCLUSION: In summary, the results of this study suggest that selegiline may be an appropriate treatment for spinal cord injuries.

Neuroprotective effect of L-deprenyl on the expression level of the Mst1 gene and inhibition of apoptosis in rat-model spinal cord injury.

Objectives: After primary tissue damage as a result of spinal cord injury (SCI), there is a period of secondary damage, which includes several cellular and inflammatory biochemical cascades. As a novel pro-apoptotic kinase, Mst1 (serine/threonine kinase 4) promotes programmed cell death in an inflammatory disease model. This study aimed to evaluate Mst1 gene expression levels in rats with spinal cord injury treated with L- deprenyl. Materials and Methods: The rats were divided into control (contusion), laminectomy, sham-operated (contused rats received 1 ml normal saline intraperitoneal), and treatment (contused rats received 5 mg/kg of L-deprenyl intraperitoneal; once a day for 7 days). The BBB (Basso, Beattie, and Bresnahan) scales were performed to assess motor function following SCI. Rats were sacrificed 28 days after SCI and the spinal cord lesion area was removed. Apoptosis and cavity formation in the spinal cord were determined by H&E staining and TUNEL assay, respectively. The mRNA levels of the Mst1, Nrf2, Bcl-2, and PGC1 α genes were analyzed using real-time quantitative PCR. Results: The results showed significant improvement in motor function in the L- deprenyl group compared with the untreated group. Histological analysis showed a significant reduction in the number of tunnel-positive cells after injection of L-deprenyl, as well as a decrease in the volume of the cavity. In addition, L-deprenyl treatment increased the expression of the Nrf2, Bcl-2, and PGC1 α genes, while reducing the expression of the Mst1 gene in the spinal nerves. Conclusion: These results suggest that L-deprenyl is a promising treatment for spinal cord injury.

Valproic Acid Ameliorates Locomotor Function in the Rat Model of Contusion via Alteration of Mst1, Bcl-2, and Nrf2 Gene Expression.

Background: In animal models of inflammatory diseases, Mammalian sterile 20-like kinase 1 (Mst1) facilitates the programmed cell death as a novel pro-apoptotic kinase. This research aimed to determine the expression level of Mst1 gene in a rat model of SCI treated with valproic acid (VPA). Methods: Severe rat model contusion was used for evaluation of the neuroprotective effect of valproic acid. The Basso-Beattie-Bresnahan test, was performed to determine locomotor functions. Hematoxylin/eosin staining and TUNEL assay were performed to detect cavity formation and apoptosis, respectively. The mRNA levels of the genes Mst1, nuclear factor (erythroid-derived 2)-like 2, and B-cell lymphoma 2 were evaluated, using quantitative real-time PCR acute spinal cord injury (RT-PCR). Results: The results revealed that Mst1 gene expression and TUNEL-positive cells in the VPA-treated group were significantly reduced as compared to the untreated group (p ≤ 0.05). Conclusion: Our findings indicate that VPA has therapeutic potential and can be a candidate for the treatment of neurodegenerative disorders and traumatic injury as a promising drug.

Involvement of Asc and Nlrp3 inflammasomes in the testes following spinal cord injury.

OBJECTIVE: The exact mechanism, by which spinal cord injury (SCI) leads to a male subfertility is not well-known. Present study was conducted to determine the mechanisms that lead to the elevated end-product cytokines and inflammasomes in the testes of an SCI rat model. Moreover, we evaluated the inflammasome components following SCI in testis over a defined time periods. METHODS: Weight drop technique was used to induce SCI at the level of the T10 vertebra in male Wistar rats. The animals were sacrificed at specific time intervals (3, 7, 14, 21, and 28 day's post-SCI). mRNA levels of inflammasomes and cytokines were measured by real-time PCR, germ cells apoptosis was evaluated by TUNEL staining, and the epithelium of seminiferous tubules by Miller's and Johnsen's scores. RESULTS: The results showed activation of Nlrp3 in the testes of SCI animals at different time points. Expression of Nlrp3 and IL-1ß sharply increased 14 days after the SCI. Upregulation of IL-1ß and IL-18 at days 14 and 21 post-SCI might disintegrate the epithelium of seminiferous tubules at day 14 and induce germ cells apoptosis, increase abnormal sperm cells, and attenuate motility and viability at 21 days post-SCI. CONCLUSION: This study provided further evidence of innate immunity activation in testes that could lead to more disruption of spermatogenesis in SCI patients at specific times.

Lovastatin alters neurotrophin expression in rat hippocampus­derived neural stem cells in vitro.

Neural stem/progenitor cells hold valuable potential for the treatment of neurodegenerative disorders. The modulation of intrinsic growth factor expression, such as neurotrophins and their receptors, is a necessary step in achieving neural stem cells (NSCs) therapy. The statins have recently been reported to provide both anti­inflammatory and neuroprotective effects. In the developing and mature nervous systems, neurotrophic factors are known to impact neuronal growth and survival. In this study, we investigated for a positive effect of lovastatin on the expression of neurotrophins in the neonatal rat hippocampus­derived NSCs. NSCs were isolated and cultured up to passage three. To confirm cellular identity, immunocytochemical evaluation and flow cytometry analysis were performed using specific antibodies. To determine the optimum concentration of lovastatin, the MTT assay was used. Neurotrophin expression was evaluated using quantitative real­time reverse transcription­polymerase chain reaction (RT­qPCR). Flow cytometry results demonstrated that NSCs were positive for nestin, a marker for neural progenitor cells. An increase in cellular viability was observed with a 24 h exposure of lovastatin. Moreover, results showed an increase in mRNA expression for all neurotrophins compared to the control group. Taken together, the results of this study add to the growing body of literature on the neuroprotective effects of statins in neurological disorders. Lovastatin is a promising therapeutic agent for the treatment of neurodegenerative disorders.

Differentiation of bone marrow derived mesenchymal stem cells into male germ-like cells in co-culture with testicular cells.

OBJECTIVE: Stem cell therapy, specifically, pre-induction of mesenchymal stem cells toward male germ-like cells may be useful in patients with azoospermia. The aim of this study was to evaluate in vitro differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs) into male germ-like cells by indirect co-culture with testicular cells in the presence of bone morphogenetic protein 4 (BMP4). METHODS: Experimental groups included: control (mouse BMSCs), treatment group-1 (BMSCs treated with BMP4), treatment group-2 (indirect co-culture of BMSCs with mouse testicular cells in the presence of BMP4) and treatment group-3 (indirect co-culture of BMSCs with testicular cells). BMSCs-derived male germ-like cells were evaluated by the expression of Dazl, and Stra8 using RT-qPCR. RESULTS: Stra8 gene expression was significantly increased in the treatment group-2 and Dazl gene was significantly increased in the treatment group-1 compared to other groups. In conclusion, indirect co-culturing of BMSCs with testicular cells and BMP4 leads to the differentiation of BMSCs into male germ-like cells which express specific male germ-like genes. Testicular cells released factors that contributed to the differentiation of BMSCs into male germ progenitor cells. CONCLUSION: This study suggests that mesenchymal stem cells may be differentiated into male germ-like cells and therefore, may be a novel treatment option for men with azoospermia.

The effect of hydroethanolic extract of Matricaria chamomilla on the reproductive system of male rats exposed to formaldehyde.

Formaldehyde is a common agent in our surrounding environment and can adversely affect the male reproductive system. In this study, the effectiveness of Matricaria chamomilla (MC) extract as an antioxidant was investigated in rats treated with formaldehyde. Thirty-two male Wistar rats were randomly divided into six groups: F (10 mg/kg formaldehyde), M200 (200 mg/kg MC extract), M500 (500 mg/kg MC extract), FM200 (10 mg/kg formaldehyde and 200 mg/kg MC extract), FM500 (10 mg/kg formaldehyde and 500 mg/kg MC extract) and control group (0.9% normal saline). Formaldehyde and MC extract were administered daily for 30 consecutive days via intraperitoneal injection. Hormonal status, sperm parameters, testis tissue histology, germinal cells apoptosis and stereological analyses of testis tissue were investigated. Testosterone and LH levels were significantly increased in FM200, FM500, F200 and F500 groups compared to F group (p ≤ 0.05). Sperm count, motility and viability were significantly enhanced in FM200, FM500, F200 and F500 groups compared to F group (p ≤ 0.05). A decrease in the number of apoptotic germ cells in FM200, FM500, M200 and M500 groups (p ≤ 0.05) was evident. In particular, the MC extract in dose 500 mg/kg is seen to reduce the adverse effects of formaldehyde on the reproductive system of male rats.

Neuroprotective effects of lovastatin in the pilocarpine rat model of epilepsy according to the expression of neurotrophic factors.

Studies have suggested that neurotrophic factors (NTFs) are involved in the status epilepticus development. This indicates their essential role in mediating acquired epileptic conditions. Therefore, modulating the expression of NTFs may inhibit seizure-induced cell loss in the epileptic lesions. In this study, we examined the anti-apoptotic, anti-necrotic and regulatory effects of lovastatin on the expression of NTFs in the pilocarpine rat model of temporal lobe epilepsy (TLE). A total of 32 male Wistar rats were divided into 4 groups (n = 8 per group): i) normal; ii) non-treated epileptic group [intraperitoneal (i.p.) administration of 350-400 mg/kg pilocarpine]; iii) treatment group (pilocarpine-treated rats treated followed by 5 mg/kg lovastatin); and iv) vehicle epileptic rats treated with Carboxymethyl cellulose (CMC). Animals that had a behavioral score of 4-5 according to the Racine scale were selected for study participation. Three days after the first seizure, pilocarpine-treated rats received i.p. injections of lovastatin for 14 days. The rats were killed and prepared for histopathologic analysis as well as real-time RT-PCR 17 days after the first seizure. The results of this study showed increased mRNA expression of glial cell line-derived neurotrophic factor (GDNF) and Ciliary neurotrophic factor (CNTF) and decreased expressions of Brain-derived neurotrophic factor (BDNF), Neurotrophin-3 (NT-3), and Neurotrophin-4 (NT-4) mRNA in the epileptic rats treated with lovastatin. Histological analysis of neurodegeneration in the brain sections showed that the number of hippocampal apoptotic and necrotic cells significantly decreased in the treatment groups. Furthermore, numerical density of neurons per area was significantly higher in the treated groups compared with the untreated groups. Collectively, the results of this study have shown that lovastatin could attenuate seizure-induced expression of neurotrophic factors and consequently reduce hippocampal cell death in the pilocarpine rat model of TLE.

The effect of lithium chloride on BDNF, NT3, and their receptor mRNA levels in the spinal contusion rat models.

OBJECTIVE: Nowadays, there seems to be no decisive way for treatment of spinal cord injury (SCI).Extensive cell death (apoptosis and necrosis) occurring in SCI can cause considerable progressive sensorimotor disabilities. Preventing cell death by improving endogenous regenerative capability could an effective strategy for the treatment of SCI. This study was designed to evaluate the effects of lithium chloride (LiCl) on the cell survival through overexpression of BDNF and NT3 mRNA level and their receptors in the contusion rat models. METHODS: Rats were randomly divided into four experimental groups (eight rats/group) including: contused animals (the non-treatment group); contused animals (the control group) which received laminectomy; contused animals received normal saline (vehicle)and contused animals received intraperitoneal injection of 20 mg/kg LiCl three days after surgery. Injection continued for 14 days as treatment. Basso, Beattie, Bresnahan (BBB) rating scale was used to assess the motor function of the rats. To evaluate the histopathological and gene expression analysis, rats were sacrificed 28 days after surgery. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed to obtain the relative levels of mRNA for BDNF, NT3 and their receptors. RESULTS: The results showed LiCl ameliorates BBB scores via up-regulation of BDNF and TrkB receptors. Also, histological analysis showed that the numerical density per area of TUNEL- positive cells and the percentage of cavity significantly decreased in the LiCl-treated group. CONCLUSION: Our findings suggest that LiCl protects neural cells and effectively enhances locomotor function, which was done through up-regulation of endogenous BDNF expression in rats with SCI. ABBREVIATIONS: SCI: spinal cord injury; LiCl: lithium chloride; BDNF: Brain-derived neurotrophic factor; NT3: Neurotrophin-3; BBB: Basso, Beattie, Bresnahan; TrkB: Tropomyosin receptor kinase B; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling.

Antioxidant and neuroprotective effects of lycopene and insulin in the hippocampus of streptozotocin-induced diabetic rats.

In the present study the antioxidant and neuroprotective effects of insulin and lycopene on passive avoidance memory, total antioxidant capacity (TAC), malondialdehyde activity (MDA) and prevention of apoptosis in the hippocampus streptozotocin-induced diabetic rats were examined. The rats were randomly divided to six experimental groups (n=8 per group): Non-diabetic (controls); diabetic; diabetic treated with lycopene; diabetic treated with insulin; diabetic treated with lycopene and insulin; and normal treated with lycopene. Intraperitoneal injection of single dose (60 mg/kg) streptozotocin (STZ) was used to induce the diabetes rat model. The shuttle box test was used for learning and memory assessment. Rats were then sacrificed and hippocampi tissue isolated from the two hemispheres to determine TAC and MDA. Apoptosis rate was also evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling and acridine orange staining assays. The results indicated that lycopene and insulin, solely or in combination, prevented hippocampal neuronal cell death and improved learning and cognition by increasing TAC and decreasing MDA. Collectively, the findings presented herein suggest that insulin and lycopene co-treatment has neuroprotective effect, and ameliorates STZ-induced learning and memory impairment and apoptotic cell death in the hippocampal regions of diabetic rats.

Indirect Co-Culture of Testicular Cells with Bone Marrow Mesenchymal Stem Cells Leads to Male Germ Cell-Specific Gene Expressions.

OBJECTIVE: Non-obstructive azoospermia is mostly irreversible. Efforts to cure this type of infertility have led to the application of stem cells in the reproduction field. In the present study, testicular cell-mediated differentiation of male germ-like cells from bone marrow-derived mesenchymal stem cells (BM-MSCs) in an in vitro indirect co-culture system is investigated. MATERIALS AND METHODS: In this experimental study, mouse BM-MSCs were isolated and cultured up to passage three. Identification of the cells was evaluated using specific surface markers by flow-cytometry technique. Four experimental groups were investigated: control, treatment with retinoic acid (RA), indirect co-culture with testicular cells, and combination of RA and indirect co-culture with testicular cells. Finally, following differentiation, the quantitative expression of germ cell-specific markers including Dazl, Piwil2 and Stra8 were evaluated by real-time polymerase chain reaction (PCR). RESULTS: Molecular analysis revealed a significant increase in Dazl expression in the indirect co-culture with testicular cells group in comparison to the control group. Quantitative expression level of Piwil2 was not significantly changed in comparison to the control group. Stra8 expression was significantly higher in RA group in comparison to other groups. CONCLUSION: Indirect co-culture of BM-MSCs in the presence of testicular cells leads to expression of male germ cell-specific gene, Dazl, in the induced cells. Combination of co-culture with testicular cells and RA did not show any positive effect on the specific gene expressions.

Effect of Ghrelin on Caspase 3 and Bcl2 Gene Expression in H2O2 Treated Rat's Bone Marrow Stromal Cells.

Purpose: The antiapoptotic effect of ghrelin in various cell lines including bone marrow stromal cells (BMSCs) has been proved. However, the real mechanism of this effect is not clear. Caspase3 and Bcl2 are well-known pro- and antiapoptotic regulatory genes in eukaryotes. The aim of the study was to find out the effect of ghrelin on Caspase 3 and Bcl2 change in BMSCs. Methods: Rat BMSCs were cultivated in DMEM. Passage 3 BMSCs were treated with ghrelin 100 µM for 48 h. Real-time PCR for Caspase 3 and Bcl2 was carried out from B (untreated BMSCs), BH (BMSCs treated with 125 µM H2O2), BGH (BMSCs treated with 100 µM ghrelin then 125 µM H2O2) and BG (BMSCs treated with 100 µM ghrelin) groups. For immunofluorescence, cells were incubated with anti Caspase 3 and Bcl2monoclonal antibodies. Primary antibodies were visualized using the FITC method. All data are presented as means ± SEM. Values of P<0.05 were considered statistically significant. Results: Ghrelin decreased mRNA expressions of Caspase-3 significantly as compared to the BH group (P<0.05). Also, Bcl-2 gene expression showed an increment in BG group as compare with BH and BGH groups (P<0.05). A high present of Bcl-2 positive cells were observed in the BGH group while Caspase-3 positive cells were significantly decreased in the BGH group compared with the BH group (P<0.05). Conclusion: Ghrelin probably enhances BMSCs viability through regulation of pro- and antiapoptotic genes Caspase 3 and Bcl2. However the signaling pathway of this effect should be elucidated in the future.

Neuroprotective effect of lovastatin through down-regulation of pro-apoptotic Mst1 gene expression in rat model pilocarpine epilepsy.

OBJECTIVE: Statins as inhibitors of HMG-CoA reductase have been recently recognized as anti-inflammatory and neuroprotective drugs. In this paper, we studied anti-apoptotic and regulatory effects of lovastatin using Pilocarpine rat model through downregulation of Mst1 (Mammalian sterile 20-like kinase 1) as a novel pro-apoptotic gene. METHODS: The rats were divided into four groups: non-treated epileptic rats, lovastatin treated, and two vehicle groups. Racine scale was used for behavioral assessment and animals with a score of 4-5 were selected for the study. After 3 days, epileptic rats received intraperitoneal injections of lovastatin, followed by treating for 14 days. Next, they were sacrificed (28 post-first seizure) and prepared for histopathological analysis and Real-time RT-PCR. RESULTS: The results showed that lovastatin protects Pilocarpine-induced cell death via a regulatory effect on pro-apoptotic and anti-apoptotic gene expression. The real-time PCR results showed that in the epileptic lovastatin treated group, the expression level of Mst1 significantly decreased while Nrf2 and Bcl-2 genes increased. Furthermore, histological analysis of neurodegeneration in the brain sections showed that the number of hippocampal apoptotic cells significantly decreased in the treatment groups. The results showed that the numerical density of neurons per area was significantly higher in the treated than the untreated group. CONCLUSION: Overall, the results of this study showed that lovastatin attenuates hippocampal cell death in Pilocarpine-induced status epilepticus rat model through downregulation of the pro-apoptotic Mst1 gene. ABBREVIATIONS: Mst1: Mammalian sterile 20-like kinase 1; Nrf2: nuclear factor erythroid 2-related factor 2; Bcl-2: B-cell lymphoma 2; HMG-CoA: 3-hydroxy-3-methylglutaryl-coenzyme A; RT-PCR: reverse transcription-polymerase chain reaction; TLE: Temporal Lobe Epilepsy; SE: status epilepticus.

Adenosine decreases oxidative stress and protects H2O2-treated neural stem cells against apoptosis through decreasing Mst1 expression.

Overproduction of free radicals during oxidative stress induces damage to key biomolecules and activates programed cell death pathways. Neuronal cell death in the nervous system leads to a number of neurodegenerative diseases. The aim of the present study was to evaluate the neuroprotective effect of adenosine on inhibition of apoptosis induced by hydrogen peroxide (H2O2) in bone marrow-derived neural stem cells (B-dNSCs), with focus on its regulatory effect on the expression of mammalian sterile 20-like kinase 1 (Mst1), as a novel proapoptotic kinase. B-dNSCs were exposed to adenosine at different doses (2, 4, 6, 8 and 10 µM) for 48 h followed by 125 µM H2O2 for 30 min. Using MTT, terminal deoxynucleotidyl transferase dUTP nick-end labeling and real-time reverse transcription polymerase chain reaction assays, the effects of adenosine on cell survival, apoptosis and Mst1, nuclear factor (erythroid-derived 2)-like 2 and B-cell lymphoma 2 and adenosine A1 receptor expression were evaluated in pretreated B-dNSCs compared with controls (cells treated with H2O2 only). Firstly, results of the MTT assay indicated 6 µM adenosine to be the most protective dose in terms of promotion of cell viability. Subsequent assays using this dosage indicated that apoptosis rate and Mst1 expression in B-dNSCs pretreated with 6 µM adenosine were significantly decreased compared with the control group. These findings suggest that adenosine protects B-dNSCs against oxidative stress-induced cell death, and therefore, that it may be used to promote the survival rate of B-dNSCs and as a candidate for the treatment of oxidative stress-mediated neurological diseases.

Ghrelin Upregulates Hoxb4 Gene Expression in Rat Bone Marrow Stromal Cells.

OBJECTIVES: Ghrelin is a peptide which has a proliferative and antiapoptotic effect in many cells including bone marrow stromal cells (BMSCs). Homeobox protein B4 (HOXB4) is a transcription factor involved in stem cell regeneration and survival. The aim of the study was to find out the efect of ghrelin on Hoxb4 expression in BMSCs. MATERIALS AND METHODS: In this experimental study, rat BMSCs were cultivated in Dulbecco's Modified Eagle Medium (DMEM). Passage three BMSCs were treated with ghrelin 100 µM for 48 hours. Real-time polymerase chain reaction (PCR) was carried out from the untreated BMSCs (B), BMSCs treated with 125 µM H2O2 (BH), BMSCs treated with 100 µM ghrelin then 125 µM H2O2 (BGH) and BMSCs treated with 100 µM ghrelin (BG) groups. For immunofluorescence, cells were incubated with an anti-HOXB4 monoclonal antibody. Primary antibodies were visualized using the Fluorescein isothiocyanate (FITC) method. All data are presented as mean ± SEM and P<0.05 was considered as statistical significant. RESULTS: Hoxb4 expression significantly increased in the BG compared with BH and BGH groups. Furthermore, 100 µM ghrelin, increased the mean of HOXB4 positive immunoreactive cells compared to the BH group. CONCLUSIONS: Ghrelin probably enhances proliferation and viability of BMSCs through Hoxb4 upregulation. However, the signaling pathway and other biological outcomes of this effect should be elucidated in different stem cells.

Neuroprotective effects of selegiline on rat neural stem cells treated with hydrogen peroxide.

Oxidative stress and reactive oxygen species generation have been implicated in the pathogenesis of several neurological disorders including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and multiple sclerosis. In the present study, the neuroprotective effects of selegiline against hydrogen peroxide-induced oxidative stress in hippocampus-derived neural stem cells (NSCs) were evaluated. NSCs isolated from neonatal Wistar rats were pretreated with different doses of selegiline for 48 h and then exposed to 125 µM H2O2 for 30 min. Using MTT and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays, acridine orange/ethidium bromide staining and reverse transcription-quantitative polymerase chain reaction, the effects of selegiline on cell survival, apoptosis and the expression of B-cell lymphoma 2 (Bcl-2) and heat shock protein 4 (Hspa4) in pretreated stem cells were assessed compared with a control group lacking pretreatment. The results indicated that the viability of cells pretreated with 20 µM selegiline was significantly increased compared with the control group (P<0.05). Additionally, 20 µM selegiline increased the mRNA expression of Bcl-2 and Hspa4 (P<0.05 vs. control) and suppressed oxidative stress-induced cell death (apoptosis and necrosis; P<0.05 vs. control and 10 µM groups). From these findings, it was concluded that selegiline may be a therapeutic candidate for the treatment of neurological diseases mediated by oxidative stress.

Di-(2-ethylhexyl) Phthalate-Induced Hippocampus-Derived Neural Stem Cells Proliferation.

The brain and spinal cord have a limited capacity for self-repair under damaged conditions. One of the best options to overcome these limitations involves the use of phytochemicals as potential therapeutic agents. In this study, we have aimed to investigate the effects of di-(2-ethylhexyl) phthalate (DEHP) on hippocampus-derived neural stem cells (NSCs) proliferation to search phytochemical candidates for possible treatment of neurological diseases using endogenous capacity. In this experimental study, neonatal rat hippocampus-derived NSCs were cultured and treated with various concentrations of DEHP (0, 100, 200, 400 and 600 µM) and Cirsium vulgare (C. vulgare) hydroethanolic extract (0, 200, 400, 600, 800 and 1000 µg/ml) for 48 hours under in vitro conditions. Cell proliferation rates and quantitative Sox2 gene expression were evaluated using MTT assay and real-time reverse transcription polymerase chain reaction (RT-PCR). We observed the highest average growth rate in the 400 µM DEHP and 800 µg/ml C. vulgare extract treated groups. Sox2 expression in the DEHP-treated NSCs significantly increased compared to the control group. Gas chromatography/mass spectrometry (GC/ MS) results demonstrated that the active ingredients that naturally occurred in the C. vulgare hydroethanolic extract were 2-ethyl-1-hexanamine, n-heptacosane, 1-cyclopentanecarboxylic acid, 1-heptadecanamine, 2,6-octadien-1-ol,2,6,10,14,18,22-tetracosahexaene, and DEHP. DEHP profoundly stimulated NSCs proliferation through Sox2 gene overexpression. These results provide and opportunity for further use of the C. vulgure phytochemicals for prevention and/or treatment of neurological diseases via phytochemical mediated-proliferation of endogenous adult NSCs.

Regulatory T cell number in multiple sclerosis patients: A meta-analysis.

Regulatory T cells (Treg cells), defined as CD4(+) CD25(+) FoxP3(+) T cells by expression of CD4, high-affinity IL-2 receptor and the transcription factor, forkhead box P3 (FoxP3). They play a pivotal role in protecting individuals from autoimmunity and a growing body of evidence suggests their role in the prevention of multiple sclerosis development. However, there are discrepancies about the type of defect in the Treg cells of multiple sclerosis patients and especially whether the Treg number alteration could be contributed to multiple sclerosis pathogenesis. Indeed, whether low number of Treg cells can be a risk factor contributing to multiple sclerosis pathogenesis is the matter of debate and there is not any comprehensive agreement on it. Thus, the objective of this systematic review and meta-analysis was to precisely quantify the nature and magnitude of the association between Treg cell number and the risk ratio/odds ratio (OR) of multiple sclerosis in the case-control studies. Hence, medical databases of Embase, PubMed/Medline, PubMed, PubMed Central and SCOPUS were searched for empirical papers using "Regulatory T cell frequency", "Treg frequency" in combination with "multiple sclerosis". In the case-control studies, papers were reviewed for inclusion/exclusion criteria and 8 publications were included. Under random-effect model meta-analysis the data showed that the frequency of Treg cells was not a risk factor in multiple sclerosis using current laboratory methods.

In vitro study of the long-term cortisol treatment effects on the growth rate and proliferation of the neural stem/precursor cells.

Adult neural stem/precursor cells (NSPCs) residing in the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus of the hippocampus are involved in the memory formations and psychological problems. It is believed that basal levels of glucocorticoids are essential for neuronal development, plasticity, and survival, while stress-mediated levels of glucocorticoids produce neuronal loss. Degeneration of NSPCs by the apoptotic and necrotic stimuli have great devastating outcomes on the brain and contributes to the pathophysiology of neurological as well as psychological disorders. Using MTT assay, acridine orange, and TUNEL assay, we have demonstrated that cortisol at high and excessive (more than 5 µM) levels had anti-proliferative effects on the NSPCs derived from subventricular and subgranular zones in a dose- and time-dependent manner through apoptosis as well as necrosis. These outcomes can highlight the role of stress-mediated decline of adult neurogenesis in the aging brain and interconnect stress-mediated cortisol secretion with brain aging diseases.