BACKGROUND: Cryopreservation of pre-antral follicles is a hopeful technique to preserve female fertility. The aim of the present study was to evaluate reactive oxygen species (ROS) and total antioxidant capacity (TAC) levels of mouse vitrified pre-antral follicles in the presence of alpha lipoic acid (ALA). METHODS: Isolated pre-antral follicles (140-150 µm in diameter) were divided into vitrified-warmed and fresh groups. Each group was subjected to in vitro maturation with or without ALA for 12 days, followed by adding human chronic gonadotropin to induce ovulation. In vitro fertilization was performed to evaluate their developmental competence. In parallel, the amount of ROS and TAC were assessed after 0, 24, 48, 72, and 96 h of culture by 2',7'-dichlorofluorescin assay and ferric reducing/antioxidant power assay, respectively. RESULTS: The respective rates of survival, antrum formation, and metaphase II oocytes were significantly higher in ALA-supplemented groups compared to the groups not treated with ALA. TAC and ROS levels were significantly decreased and increased, respectively during the culture period up to 96 h in the absence of ALA in both vitrified and non-vitrified samples. However, with pretreatment of ALA, TAC levels were increased significantly and remained constant up to 96 h in vitrified-warmed pre-antral follicles, while ROS levels completely returned to the level of starting point after 96 h of culture in the presence of ALA. CONCLUSION: Pretreatment of ALA positively influences development of pre-antral follicles in vitrified and non-vitrified samples through increasing follicular TAC level and decreasing ROS levels.
Objective(s): Stem cell therapy is believed to be as a promising treatment strategy for tissue repair and regeneration. The plasticity specification of the adult stem cells, such as MSCs, has enabled that these cells to be used in the treatment of a broad spectrum of diseases like liver disorders. In this study, the production of urea and Albumin (Alb), glycogen storage, and expression of some liver genes including α-fetoprotein (AFP), Alb, cytokeratin18 (CK18) and cytokeratin19 (CK19) was compared between mesenchymal stem cells (MSCs) and isolated rat hepatocytes. Materials and Methods: The MSCs were isolated from rat femurs and tibias and cultured in α-MEM, DMEM and RPMI mediums supplemented with serum. Hepatocytes were isolated from Rat livers and cultured in DMEM with serum. The expression of AFP, Alb, CK18, and CK19 genes was evaluated using the reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, the synthesis of albumin and urea of the cells was measured. Results: In vitro conditions, MSCs and hepatocytes exhibited the characteristic functions of the liver such as capacity to synthesize Alb, urea, the storage of glycogen. In this study, the expression of some liver genes such as AFP, Alb, CK18 and CK19 at mRNA levels was also shown. Conclusion: The results showed that MSCs exhibited some liver functions, and may be considered as an alternative source for adult stem cell transplantation in liver repair due to the excellent proliferation and differentiation capacities.
Introduction: Mesenchymal stem sells (MSCs) have an important role in cell therapy due to those high proliferation, differentiation and cell regeneration capacity. Advent signs of Replicative Cellular Senescence (R.C.S) in culture condition are one of the problems in stem cells culture pathway before treatment. R.C.S is associated with reducing the capacity of antioxidant enzymes and matrix metalloproteinase (MMPs) proteins changes. The aim of this study was to areinvestigate and compare the production capacity of antioxidant superoxiddismutase (SOD) and glutathione peroxidase (GPx) enzymes and matrix metalloproteinases (MMP-2) in conditional medium of rat bone marrow mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ADSCs). Materials and Methods: BMSCs and ADSCs were respectively isolated from the tibia and femur bones and subcutaneous adipose tissues. These cells were cultured for alternative 7 passages in α-MEM supplemented with 10% fetal bovine serum. Evaluation of antioxidant and MMP activity in the conditional medium without serum was respectively assayed by enzymatic assay and zymography. Results: Two types of cells studied almost identical have most activity in the expression of SOD and GPx, respectively, in 5 and 4-6 passages. Also, demonstrated MMP activity is highest in the fourth passage. On the other hand, in these cells, membrane health reported with measurement of MDA and LDH. Conclusion: Adipose tissue has a population of stem cells with high capacity of antioxidant. Also, BMSCs and ADSCs are at highest level of antioxidant and MMP expression capacity in 4-6 passages.
Introduction: The reduction of dopamine level caused by neurodegenerative diseases such as Parkinson's disease (PD) may reduce the production of new neurons in dentate gyrus (DG) of the hippocampus. In addition, there is a direct link between the reduction of neurons in the hippocampus and memory impairment. In this study, the effect of carnosic acid (CA) on the hippocampal neurogenesis was evaluated after 6-OHDA injury. Materials and Methods: Male Wistar rats were randomly divided into six groups. First group was injected by bilateral intra-nigral of 6-OHDA at a dose of 6μg (injury). Groups 2-5 were injured rats which received orally Rosemary extract containing 40% CA at doses of 25, 50 and100 mg/kg (treated) and distilled water (control), once daily in a period of 14 days before and after injury. The sixth group (sham) was injected with saline instead of neurotoxin. After treatment, the brains were removed and fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin and cut into 10μm thick slices. Sections were stained with cresyl fast violet and cell counting of hippocampal regions was done.The loss of dopaminergic neurons in the 6-hydroxydopamine-lesioned rats, compared to sham-operated rats, was verified by tyrosine hydroxylase immunohistochemistry. Results: Immunostaining analysis revealed a high density of TH+ cells in sham compared to injured group. The number of DG granular and CA1 pyramidal cells were decreased significantly in both control and injured groups compared to sham (P<0.05). The number of granular and CA1pyramidal cells were increased significantly in CA (25, 50,100) and CA (50,100) treated groups respectively, compared to control and injured rats (P<0.05). The number of CA3 pyramidal cells was not increased significantly in treated groups compared to control and injured rats. Conclusion: CA plays an important role in protecting hippocampal neurons from further damage in response to 6-OHDA. Then it is the effective herbal drug with treatment potential to improve memory impairment in PD which caused by neurons degeneration in the hippocampus.
AUTHOR KEYWORDS: 6-Hydroxydopamine; Carnosic acid; Dopamine; Hippocampus; Neuroprotective INDEX KEYWORDS: carnosic acid; oxidopamine; Rosmarinus officinalis extract, animal experiment; animal model; animal tissue; article; brain injury; controlled study; hippocampal CA1 region; immunohistochemistry; male; memory disorder; nervous system development; neuroprotection; nonhuman; pyramidal nerve cell; rat PUBLISHER: Semnan University of Medical Sciences
Background: Cryopreservation of pre-antral follicles is a hopeful technique to preserve female fertility. The aim of the present study was to evaluate reactive oxygen species (ROS) and total antioxidant capacity (TAC) levels of mouse vitrified pre-antral follicles in the presence of alpha lipoic acid (ALA). Methods: Isolated pre-antral follicles (140-150 μm in diameter) were divided into vitrified-warmed and fresh groups. Each group was subjected to in vitro maturation with or without ALA for 12 days, followed by adding human chronic gonadotropin to induce ovulation. In vitro fertilization was performed to evaluate their developmental competence. In parallel, the amount of ROS and TAC were assessed after 0, 24, 48, 72, and 96 h of culture by 2',7'-dichlorofluorescin assay and ferric reducing/antioxidant power assay, respectively. Results: The respective rates of survival, antrum formation, and metaphase II oocytes were significantly higher in ALA-supplemented groups compared to the groups not treated with ALA. TAC and ROS levels were significantly decreased and increased, respectively during the culture period up to 96 h in the absence of ALA in both vitrified and non-vitrified samples. However, with pretreatment of ALA, TAC levels were increased significantly and remained constant up to 96 h in vitrified-warmed pre-antral follicles, while ROS levels completely returned to the level of starting point after 96 h of culture in the presence of ALA. Conclusion: Pretreatment of ALA positively influences development of pre-antral follicles in vitrified and nonvitrified samples through increasing follicular TAC level and decreasing ROS levels.
Background: In spite of extensive efforts to improve in vitro oocyte maturation, the obtained results are not very efficient. This study was conducted to assess impacts of cAMP elevating agents and alpha lipoic acid (ALA) on in vitro oocyte maturation and fertilization. Methods: Mouse germinal vesicle (GV) oocytes were categorized into cumulus denuded oocytes (DOs; n=896) and cumulus oocyte complexes (COCs; n=1077) groups. GV oocytes were matured in vitro with or without ALA; (I) without the meiotic inhibitors; (II) supplemented with cilostamide; (III) supplemented with forskolin and (IV) supplemented with Forskolin plus cilostamide. The obtained metaphase II (MII) oocytes were subjected to in vitro fertilization. Independent-samples t-testand ANOVA were used for data analysis. A p-value less than 0.05 was considered to be statistically significant. Results: The COCs maturation, fertilization and two cell embryo rates were higher than those of DOs in the control group, while no significant difference was observed between relevant COCs and DOs when they were cultured with cilostamide meiotic inhibitors in two step manner. Combined treatment of cilostamide and forskolin significantly elevated the developmental rates in both COCs and DOs as compared to other groups. The developmental rates of COCs and DOs in the presence of ALA were similar to their respective groups without ALA. Conclusion: cAMP elevating agents were more effective on DOs than COCs with regard to rates of maturation and fertilization. However, ALA did not affect the developmental rates of both COCs and DOs in in vitro maturation in one or two step manner.
AUTHOR KEYWORDS: ALA; cAMP-elevating agents; Cumulus cell; In vitro maturation; Mouse; Oocyte INDEX KEYWORDS: cilostamide; cyclic AMP; forskolin; thioctic acid, animal cell; animal experiment; article; controlled study; cumulus cell; drug effect; embryo; embryo culture; female; germinal vesicle; in vitro oocyte maturation; male; mouse; nonhuman; oocyte maturation; spermatozoon; spermatozoon density
Objective: There is longstanding experimental and clinical evidence that supports the idea that replacement of dopaminergic (DAergic) neurons can ameliorate functional disabilities of Parkinson's disease (PD). The purpose of the present study is to examine the efficacy of transplantation of rat bone marrow stromal cell (BMSCs)-derived tyrosine hydroxylase-positive (TH +) cells induced by deprenyl into 6-hydroxydopamine (6-OHDA)-lesioned rat models, using behavioral tests and immunohistochemical evaluations. Materials and Methods: In this experimental study, undifferentiated BrdU-labeled BMSCs were incubated in serum-free medium that contained 10-8 M deprenyl for 24 hours. Afterwards, BMSCs were cultured for 48 hours in α-minimal essential medium (α-MEM) supplemented with 10% FBS, then differentiated into TH+ neurons. We randomly divided 24 hemiparkinsonian rats as follows: group 1 (control) received only medium, while groups 2 and 3 were injected with 2×105 BMSCs and deprenyl-treated cells in 4 μl medium. Injections were made into the injured strata of the rats. Rotational behavior in response to apomorphine was tested before transplantation and at 2, 4, and 6 weeks post-graft. Animals were then sacrificed, and the brains were extracted for immunohistochemical and electron microscopic studies. Results: Apomorphine-induced rotation analysis indicated that animals with grafted cells in groups 2 and 3 exhibited significantly less rotational behavior than those in the control group at 2, 4, and 6 weeks after transplantation. Immunohistochemical analysis demonstrated that BrdU-labeled cells expressed specific neuronal markers, such as NF 200 and TH, at the implantation site. The presence of TH+ cells in conjunction with the reduction in rotation might show the capacity of grafted cells to release dopamine. Ultrastructural analysis revealed the presence of immature neurons and astrocyte-like cells at the graft site. Conclusion: TH+ neurons induced by deprenyl can be considered as a cell source for PD autograft therapy.
Objective: It has been reported that rat bone marrow stromal cells (BMSCs) can be spontaneously differentiated into neural-like cells without any supplemental growth factors and/or chemical treatment after long-term culture.This study aims to determineWhether, growth factors secreted by MSCs could induce self-differentiation into neural-like cells in a long-term culture. Materials and Methods: This study consisted of two groups: i. rat BMSCs (passage 5) were cultured in alfa- minimal essential medium (α-MEM) and 10% fetal bovine serum (FBS) without the addition of inducer and exchanging medium for three weeks, as the experimental group and ii.rat BMSCs (passage 5) as the control group. Each group was analysed by reverse transcriptase polymerase chain reaction (RT-PCR) to evaluate the expressions of neurotrophic factors and neural marker genes. Statistical analyses were carried out using one-way analysis of variance (ANOVA) and Tukey's multiple comparison with SPSS software (version 16). P< 0.05 was considered statistically significant. Results: The experimental group (fifth passage of BMSCs) obtained from adult rats spontaneously differentiated into neural precursor cells after long-term culture. Cultured cells expressed tyrosine hydroxylase (TH), Nurr1 and nestin genes. Furthermore, some growing cells in suspension became neurosphere-like. Self-differentiated rat MSCs (SDrMSCs) expressed significantly higher levels of NGF (0.96 ± 0.16), nestin (0.63 ± 0.08), and Nurr1 (0.80 ± 0.10) genes (p<0.05). Conclusion: In this study, we reported that rMSCs in long-term culture underwent spontaneous transformation to neural precursors without the supplement of growth factors and specific chemicals. Cells expressed neural markers such as: TH, Nurr1, and nestin genes.
Objective: This research study is an attempt to examine whether the administration of ethanol after memory reactivation would modulate subsequent expression of memory in rats. Additionally, we examined whether this administration alters the density of Cornu Ammonis (CA)1 and CA3 pyramidal and dentate gyrus (DG) granule cells. Materials and Methods: In this experimental study, adult male Wistar rats (200-300 g) were trained in a fear conditioning system using two 1 second, 0.6 mA shocks with an interval of 180 seconds. Twenty four hours later rats were returned to the chamber for 120 seconds. Immediately after reactivation they were injected with ethanol (0.5, 1, 1.5 mg/kg) or saline. 1, 7 and 14 days after reactivation, rats were returned to the context for 5 minutes. Seconds of freezing (absence of all movement except respiration) were scored. In the second experiment (described in the previous paragraph), after test 1, animals were anesthetized with sodium pentobarbital and perfused transcardially with phosphate buffer (10 minutes) and 4% paraformaldehyde (15 minutes). The brains were postfixed in phosphate-buffered 4% paraformaldehyde (24 hours) and 30% sucrose. 10-μm sections were stained with cresyl violet. Data were analyzed by 1-and 2-way ANOVA for repeated measurements by means of SPSS 16.0. Tukey's post hoc test was performed to determine the source of detected significant differences. P <0.05 were considered significant. Data are presented as mean ± SEM. Results: Findings from the first experiment indicated that ethanol at a dose of 1.5 mg/kg significantly impaired recall of memory only in the first test. The density of CA1 and CA3 pyramidal and DG granule cells in the ethanol group was decreased (p< 0.01) compared with control group respectively 43.7%, 35.8%, and 37.8. Conclusion: The data demonstrate that ethanol exposure impairs post retrieval processes. Moreover, ethanol decreases the density of CA1, CA3 and DG cells. Presumably it would be a correlation between our behavioral and histological results.
Objective(s): The purpose of this study was to investigate the role of oxidative stress in Purkinje cell neurotoxicity of ethanol-treated rat. Materials and Methods: Male rat pups 4-day-old was used in this study. Ethanol was administered to rat pups at a dose of 6 g/kg from postnatal days (PDs) 4 to 5. Pups were killed 90 min after the second alcohol treatment on PD 5 by decapitation and the brain was immediately removed. The cerebellum was dissected for analyzing the oxidative stress parameters and histological study. The activities of several antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in vermis of cerebellum were assayed. Thiobarbituric acid reactive substances (TBARS) levels were also measured as a marker of lipid peroxidation. Results: Administration of ethanol significantly increased TBARS levels in the cerebellum compared to control pups (P< 0.01). The treated pups with ethanol exhibited a marked decrease in the GPx activity (P< 0.01) whereas, in spite of decrease in the activities of SOD and CAT, when compared to control, there were not significant differences. The spherical cell bodies of Purkinje cells in control rats are aligned nicely between the granular and molecular layers. In ethanol treated pups, Purkinje cells scattered within the Purkinje cell layer and shrinkage of the cell somata is seen. Conclusion: The results of the present work demonstrated that ethanol exposure during the vulnerable window could increase TBARS levels (lipid peroxidation) and decrease GPx levels in pup's cerebellum. Also, the results confirmed ethanol-induced microencephaly, cerebellar Purkinje cell loss. These findings suggest that Purkinje cell loss is, in part through decrease in the activity of GPx and increase of lipid peroxidation in the rat cerebellum.
Objective: This study is an attempt to examine the transdifferentiation of bone marrow stromal cells (BMSCs) into tyrosine hydroxylase immunoreactive cells in parkinsonian rats associated with angiogenesis. Materials and Methods: In this study, Sprague-Dawley rats received unilateral stereotaxic injections of 6-hydroxydopamine(6-OHDA) into the left corpus striatum and then were divided into two groups. One group, the negative control, received only medium while the other group was treated with BMSCs. BMSCs were harvested from femur bones, labeled with bromodeoxyuridine (BrdU) and then transplanted into parkinsonian rats, where a behavioral study and immunohistochemistry were used to evaluate the treatment. Results: The results showed statistically significant improvement in rotational behavior. Anti-BrdU antibody showed engraftment of the transplanted cells at the transplantation site. Additionally, double immunolabeling confirmed that these cells were positive for neurofilament-200 and tyrosine hydroxylase (TH). Conclusion: It may be concluded that BMSCs transplants could engraft and differentiate into TH immunoreactive cells which may cause recovery from motor deficits. Also, BMSCs may contribute to angiogenesis at the transplantation site.
Objective: Two types of stem cells are found in the bone marrow: hematopoietic stem cells and marrow stromal cells (MSCs). Is it possible to induce the differentiation of bone marrow stromal cells into neural cells in vitro and subsequently transplant them into the brain? This might help repair neural lesions observed in some neurodegenerative diso ders such as Parkinson's disease (PD). Materials and Methods: In this study, cultured MSCs were incubated in serum free medium containing 10-8 M selegiline for 24 hours and cells were cultured for another 48 hours in áminimal essential medium (α-MEM) containing 20% fetal bovine serum (FBS). Then selegiline-treated cells were immunostained for neuronal markers such as NF-200 and TH. Results: Cell counting results showed that Selegiline at doses of 10-8, 10-7 and 10-8 M increased the mean percent of viable cells. The most effective dose of Selegiline for diferentiation of bone marrow stromal cells (BMSCs) was 10-8 M. Molecular studies indcated that the expression of BDNF, GDNF, NGF, NT3, and NT4/5 genes were increased in Selegiline-treated cells compared to non-treated group. Conclusion: BMSCs can be directed to a neural fate in vitro and can be considered as a cell source in neurological disorders for autograft therapy.
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