Metformin therapy attenuates pro-inflammatory Microglia by inhibiting NF-κB in cuprizone demyelinating mouse model of multiple Sclerosis

Multiple sclerosis (MS) is a chronic disorder characterized by reactive gliosis, inflammation, and demyelination. Microglia plays a crucial role in the pathogenesis of MS and has the dynamic plasticity to polarize between pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. Metformin, a glucose-lowering drug, attenuates inflammatory responses by activating adenosine monophosphate protein kinase (AMPK) which suppresses nuclear factor kappa B (NF-κB). In this study, we indirectly investigated whether metformin therapy would regulate microglia activity in the cuprizone (CPZ)-induced demyelination mouse model of MS via measuring the markers associated with pro- and anti-inflammatory microglia. Evaluation of myelin by luxol fast blue staining revealed that metformin treatment (CPZ + Met) diminished demyelination, in comparison to CPZ mice. In addition, metformin therapy significantly alleviated reactive microgliosis and astrogliosis in the corpus callosum, as measured by Iba-1 and GFAP staining. Moreover, metformin treatment significantly downregulated the expression of pro-inflammatory associated genes (iNOS, H2-Aa, and TNF-α) in the corpus callosum, whereas expression of anti-inflammatory markers (Arg1, Mrc1, and IL10) was not promoted, compared to CPZ mice. Furthermore, protein levels of iNOS (pro-inflammatory marker) were significantly decreased in the metformin group, while those of Trem2 (anti-inflammatory marker) were increased. In addition, metformin significantly increased AMPK activation in CPZ mice. Finally, metformin administration significantly reduced the activation level of NF-κB in CPZ mice. In summary, our data revealed that metformin attenuated pro-inflammatory microglia markers through suppressing NF-κB activity. The positive effects of metformin on microglia and remyelination suggest that it could be used as a promising candidate to lessen the incidence of inflammatory neurodegenerative diseases such as MS

A Case Based-Shared Teaching Approach in Undergraduate Medical Curriculum: A Way for Integration in Basic and Clinical Sciences

To present a multiple-instructor, active-learning strategy in the undergraduate medical curriculum. This educational research is a descriptive one. Shared teaching sessions, were designed for undergraduate medical students in six organ-system based courses. Sessions that involved in-class discussions of integrated clinical cases were designed implemented and moderated by at least 3 faculties (clinicians and basic scientists). The participants in this study include the basic sciences medical students of The Tehran University of Medical Sciences. Students’ reactions were assessed using an immediate post-session evaluation form on a 5-point Likert scale. Six two-hour sessions for 2 cohorts of students, 2013 and 2014 medical students during their two first years of study were implemented from April 2014 to March 2015. 17 faculty members participated in the program, 21 cases were designed, and participation average was 60 % at 6 sessions. Students were highly appreciative of this strategy. The majority of students in each course strongly agreed that this learning practice positively contributed to their learning (78%) and provided better understanding and application of the material learned in an integrated classroom course (74%). They believed that the sessions affected their view about medicine (73%), and should be continued in future courses (80%). The percentage demonstrates the average of all courses. The program helped the students learn how to apply basic sciences concepts to clinical medicine. Evaluation of the program indicated that students found the sessions beneficial to their learning

In vitro maturation media, cysteamine concentration and glutathione level affect blstocysts development in mouse

Background: Preparation of oocytes is one of the critical factors that determine the developmental competence of embryos produced by in vitro fertilization (IVF). Objective: In this study, the effect of cysteamine, type of media and glutathione (GSH) level on blastocysts development after in vitro maturation of mouse oocytes were investigated. Materials and Methods: Premature female mice were primed with pregnant mare stimulating gonadotrophin (PMSG), and germinal vesicle (GV) stage oocytes were obtained 45 hr later. GV oocytes were cultured in presence of 0, 50, 100, 200 and 500 µm cysteamine in TCM199 and MEME media. After IVM, MII oocytes were in vitro fertilized (IVF) and in vitro cultured (IVC) in order to observe embryo development. A group of In Vivo Ovulated (IVO) oocytes after priming with PMSG and HCG also were included in this study. 5,5-Dithio-bis (2nitrobenzoic acid) DTNB-recycling protocol was used for GSH assay. Results: Rate of IVM and IVF were improved in all oocytes treated with cysteamine in the two medium except 500 µm (81% MII rate in TCM and 64% MII in MEME). Rate of blastocyst in 100 µm cysteamine in TCM1199 and 200 µm in MEME was higher compared to control groups (In TCM 45% and in MEME 35%). In vivo MII and GV oocytes represented the highest and lowest GSH level respectively. Conclusion: Our results revealed that the media and concentration of cysteamine can affects on IVM, IVF and rate of blastocysts development on dose dependant manner

Comparison of differentiation potential of male mouse adipose tissue and bone marrow derived-mesenchymal stem cells into germ cells

Background: Recent publications about differentiation of stem cells to germ cells have motivated researchers to make new approaches to infertility. In vitro production of germ cells improves understanding differentiation process of male and female germ cells. Due to the problem of using embryonic stem cells (ESC), it’s necessary the mentioned cells be replaced with some adult multi-potent stem cells in laboratories. Objective: The aim of this study was to obtain germ cells from appropriate source beyond ESC and compare differential potentials of adipocytes derived stem cells (ADMSCs) with bone marrow derived stem cells (BMMSCs). Materials and Methods: To find multi-potential entity, after providing purified ADMSCs and BMMSCs, differentiation to osteoblast and adipocyte was confirmed by using appropriate culture medium. To confirm mesenchymal lineage production superficial markers (expression of CD90 and CD44 and non-expression of CD45 and CD31) were investigated by flowcytometry. Then the cells were differentiated to germ cells in inductive medium containing retinoic acid for 7days. To evaluate germ cells characteristic markers [Dazl (Deleted in azoospermia-like), Mvh (Mouse vasa homolog gene), Stra8 (Stimulated by retinoic acid) and Scp3 (Synaptonemal complex protein 3)] flowcytometry, imunoflorescence and real time PCR were used. Results: Both types of cells were able to differentiate into osteoblast and adipocyte cells and presentation of stem cell superficial markers (CD90, CD44) and absence of endothelial and blood cell markers (CD31, CD45) were confirmative The flowcytometry, imunoflorescence and real time PCR results showed remarkable expression of germ cells characteristic markers (Mvh, Dazl, Stra8, and Scp3). Conclusion: It was found that although ADMSCs were attained easier and also cultured and differentiated rapidly, germ cell markers were expressed in BMMSCs significantly more than ADMSCs

The effects of cumulus cells on in vitro maturation of mouse germinal vesicle stage oocytes

Background: In vitro maturation (IVM) of oocytes is a promising technique to reduce the costs and avert the side-effects of gonadotropin stimulation for in vitro fertilization (IVF). The pregnancy rates from oocytes matured in vitroare much lower than those of in vivo stimulation cycles, indicating that optimization of IVM remains a challenge. Objective: In this study, we investigated the effect of cumulus cells on maturation and fertilization rate of immature oocytes (Germinal vesicle). Materials and Methods: Germinal vesicle (GV) oocytes were recovered from 6-8 weeks old Balb C female mice 48hr after injection of 10 IU pregnant mare serum gonadotropin (PMSG). Collected oocytes were divided into two groups. Group A: GV oocytes without cumulus (denuded oocyte). Group B: GV oocytes with cumulus cells (cumulus-oocyte complex). The oocytes in both groups were cultured inTCM-199 medium in a humidified atmosphere of 5% CO2 in air at 37ºC. The maturation, fertilization and developmental rates were recorded after 24hr. Results: Maturation, fertilization and developmental rates in denuded oocytes (DO) were 65.1%, 68.02%, 78.63% respectively, and in cumulus-oocyte complex (COC) were 78.20%, 85.57% and 85.05%, respectively. The maturation, fertilization and developmental rates of COC were significantly higher than those of DO (p<0.05). Conclusion: The results show that cumulus cells have beneficial effects on maturation, fertilization and cleavage rates of mice oocytes

Progesterone Enhanced Remyelination in the Mouse Corpus Callosum After Cuprizone Induced Demyelination

Background: Progesterone as a sex steroid hormone is thought to affect and prevent demyelination, but its role in promoting myelin repair is far less investigated. In this study, remyelinating potential of progesterone in corpus callosum was evaluated on an experimental model of MS. Methods: In this experimental study, adult male C57BL/6 mice were fed with 0.2% (w/w) cuprizone in ground breeder chow ad libitum for 6 weeks. At day zero, after cuprizone removal, mice were divided randomly into two groups: (a) placebo group, which received saline pellet implant, (b) progesterone group, which received progesterone pellet implant. Some mice of the same age were fed with their normal diet to serve as the healthy control group. Two weeks after progesterone administration, Myelin content was assessed by Luxol-fast blue staining. The myelin basic protein (MBP) and proteolipid protein (PLP) expression were assessed using Western blot analysis and the changes in the number of oligodendrocytes and oligodendroglial progenitor cells were assessed by immunohistochemistry (IHC) and flow cytometry. Results: Luxol-fast blue staining revealed enhanced remyelination in the progesterone group when compared with the placebo group. Densitometry measurements of immunoblots demonstrated that MBP and PLP proteins contents were significantly increased in the progesterone group compared with the placebo group. Flow cytometry and IHC analysis showed increases in Olig2 and O4 cells in the progesterone group compared with the placebo group. Conclusion: Overall, our results indicate that progesterone treatment can stimulate myelin production and that it may provide a feasible and practical way for remyelination in diseases such as multiple sclerosis

Effects of Different Doses of Hyaloronan on Human Sperm Motility, Vitality and Morphology

Important aspect of sperm function such as motility and capacitation appear to be mediated at least partially though hyaloronic acid (HA). Present study investigated effects of different doses of HA on sperm motility and vitality in human. Sperm was obtained from 20 male from IVF clinic in Imam Khomeini Hospital. Sperm motility and vitality in human semen was analyzed according to WHO criteria before and 4 hours after treatment with different doses of HA (0.750, 1000 and 1250 &amp;micro;g/ml). The results showed that in 1000 &amp;micro;g/ml the percent of stage 3 and 4 increased compare to control group. Percent of stage 1 and 2 decreased in group with 1000 &amp;micro;g/ml HA, there was an increase in the percentage of stage 3 and 4 and decrease in percentage of stage 1 and 2 compare to control. In the group treated with 1250 &amp;micro;g/ml stage 1 and 2 increased while stage 3 and 4 decreased. Vitality in all groups decreased except of the group treated with 1000 &amp;micro;g/ml HA. The group with 1250 &amp;micro;g/ml showed significantly decrease in vitality compare to fresh group (P &amp;lt; 0.05). The present study showed that the effects of HA on sperm motility and vitality is dose dependant and 1000 &amp;micro;g/ml HA had the effective role on sperm parameters

Protective effects of erythropoietin against cuprizone-induced oxidative stress and demyelination in the mouse corpus callosum

Objective(s): Increasing evidence in both experimental and clinical studies suggests that oxidative stress plays a major role in the pathogenesis of multiple sclerosis. The aim of the present work is to investigate the protective effects of erythropoietin against cuprizone-induced oxidative stress. Materials and Methods: Adult male C57BL/6J mice were fed a chow containing 0.2 % cuprizone for 6 weeks. After 3 weeks, mice were simultaneously treated with erythropoietin (5,000 IU/ kg body weight) by daily intraperitoneal injections. Results: Our results showed that cuprizone induced oxidative stress accompanied with down-regulation of subunits of the respiratory chain complex and demyelination of corpus callosum. Erythropoietin antagonized these effects. Biochemical analysis showed that oxidative stress induced by cuprizone was regulated by erythropoietin. Similarly, erythropoietin induced the expression of subunits of the respiratory chain complex over normal control values reflecting a mechanism to compensate cuprizone-mediated down-regulation of these genes. Conclusion: The data implicate that erythropoietin abolishes destructive cuprizone effects in the corpus callosum by decreasing oxidative stress and restoring mitochondrial respiratory enzyme activity