Combination Therapy of Mesenchymal Stem Cell Transplantation and Astrocyte Ablation Improve Remyelination in a Cuprizone-Induced Demyelination Mouse Model.

Astrocytes display an active, dual, and controversial role in multiple sclerosis (MS), a chronic inflammatory demyelination disorder. However, mesenchymal stem cells (MSCs) can affect myelination in demyelinating disorders. This study aimed to investigate the effect of single and combination therapies of astrocyte ablation and MSC transplantation on remyelination in the cuprizone (CPZ) model of MS. C57BL/6 mice were fed 0.2% CPZ diet for 12 weeks. Astrocytes were ablated twice by L-a-aminoadipate (L-AAA) at the beginning of weeks 13 and 14 whereas MSCs were injected in the corpus callosum at the beginning of week 13. Motor coordination and balance were assessed through rotarod test whereas myelin content was evaluated by Luxol-fast blue (LFB) staining and transmission electron microscopy (TEM). Glial cells were assessed by immunofluorescence staining while mRNA expression was evaluated by quantitative real-time PCR. Combination treatment of ablation of astrocytes and MSC transplantation (CPZ + MSC + L-AAA) significantly decreased motor coordination deficits better than single treatments (CPZ + MSCs or CPZ + L-AAA), in comparison to CPZ mice. In addition, L-AAA and MSCs treatment significantly enhanced remyelination compared to CPZ group. Moreover, combination therapy caused a significant decrease in the number of GFAP+ and Iba-1+ cells, whereas oligodendrocytes were significantly increased in comparison to CPZ mice. Finally, MSC administration resulted in a significant upregulation of BDNF and NGF mRNA expression levels. Our data indicate that transient ablation of astrocytes along with MSCs treatment improve remyelination through enhancing oligodendrocytes and attenuating gliosis in a chronic demyelinating mouse model of MS.

Glial Response to Intranasal Mesenchymal Stem Cells in Intermittent Cuprizone Model of Demyelination.

Intranasal mesenchymal stem cells (MSCs) delivery is a non-invasive method that has received interests for treatment of neurodegenerative diseases, such as multiple sclerosis (MS). The impact of intranasal MSCs on intermittent cuprizone model of demyelination was a focus of this study. C57/BL6 mice were fed with 0.3% cuprizone in an intermittent or single ways. Luxol fast blue (LFB), Rotarod test, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry and western blot (WB) were used for interpretation of outcomes. MSCs effectively homed to the corpus callosum area, were able to improve motor coordination and to promote myelin recovery in the intermittent cuprizone (INTRCPZ/MSCs). Astrogliosis (GFAP+ cells) and microgliosis (Iba-1+ cells) were hampered, and more mature oligodendrocyte cells (APC+ cells) were identified in mice receiving INTRCPZ/MSCs. Such treatment also considerably reduced markers related to the macrophage type 1 (M1) cells, namely iNOS and CD86, but it recovered the M2 markers MRC-1 and TREM-2. In addition, a remarkable decrease in the expressions of pro-inflammatory IL-1ß and TNFα but an increase in the rate of anti-inflammatory TGF-ß and IL-10 were identified in mice that underwent INTRCPZ/MSCs therapy. Finally, microvascular changes were evaluated, and a noticeable increase in the expression of the endothelial cell marker CD31 was found in the INTRCPZ/MSCs-treated mice (p < 0.05 for all). The outcomes are representative of the efficacy of intranasal MSCs delivery in intermittent cuprizone model of MS for reshaping macrophage polarity along with modification of glial, inflammatory, and angiogenic markers in favor of therapy.

The effects of mesenchymal stem cells transplantation on A1 neurotoxic reactive astrocyte and demyelination in the cuprizone model.

Multiple sclerosis (MS), which is an autoimmune disease, is characterized by symptoms such as demyelination, axonal damage, and astrogliosis. As the most abundant type of glial cells, astrocytes play an important role in MS pathogenesis. Mesenchymal stem cells (MSCs) are a subset of stromal cells that have the potential for migration, immune-modulation, differentiation, remyelination, and neuroregeneration. Therefore, the present study evaluates the effects of MSC transplantation on A1 reactive astrocytes and the remyelination process in the cuprizone mouse model. The study used 30 male C57BL/6 mice, which were randomly distributed into three subgroups (n = 10), i.e., control, cuprizone, and transplanted MSCs groups. In order to generate a chronic demyelination model, the mice in the cuprizone group received food mixed with 0.2% cuprizone powder for 12 weeks. Then, 2 µl of DMEM containing approximately 3 × 105 DiI labeled cells was injected with a 4-min interval into the right lateral ventricle using a 10-µl Hamilton syringe. After 2 weeks of cell transplantation, we used the rotarod test to evaluate the behavioral deficits, while the remyelination process was assessed by transmission electron microscopy (TEM) and Luxol Fast Blue (LFB) staining. We assessed the population of A1 astrocytes and oligodendrocytes using specific markers, such as C3, GFAP, and Olig2, using the immunefleurocent method. The pro-inflammatory and trophic factors were assessed by a real-time polymerase chain reaction. According to our data, the specific marker of A1 astrocytes (C3) decreased in the MSCs group, while the number of oligodendrocytes significantly increased in this group compared to the cuprizone mice. Additionally, MSC was able to enhance the remyelination process after cuprizone usage, as shown by LFB and TEM images. The molecular results showed that MSCs could reduce pro-inflammatory factors, such as IL-1 and TNF-α, through the secretion of BDNF and TGF-ß as trophic factors. The obtained results indicated that MSC could reduce demyelination and inflammation by decreasing A1 neurotoxic reactive astrocytes and neurotrophic and immunomodulatory factors secretion in the chronic cuprizone demyelination model.

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.

Effect of CSF1R inhibitor on glial cells population and remyelination in the cuprizone model.

Multiple sclerosis is a kind of autoimmune and demyelinating disease with pathological symptoms such as inflammation, myelin loss, astrocytosis, and microgliosis. The colony stimulating factor 1 receptor (CSF1R) is an essential factor for the microglial function, and PLX3397 (PLX) is its specific inhibitor. In this wstudy, we assessed the effect of different doses of PLX for microglial ablation on glial cell population and remyelination process. Sixty male C57BL/6 mice (8 weeks old) were divided into 6 groups. The animals were fed with 0.2% cuprizone diet for 12 weeks. For microglial ablation, PLX (290 mg/kg) was added to the animal food for 3, 7, 14 and 21 days. Glial cell population was measured using immunohistochemistry. The rate of remyelination was evaluated using electron microscopy and Luxol Fast Blue staining. The expression levels of all genes were assessed by qRT-PCR method. Data were analysed using GraphPad Prism and SPSS software. The results showed that the administration of different doses of PLX significantly reduced microglial cells (p ≤ .001). PLX administration also significantly increased oligodendrocytes population (p ≤ .001) and remyelination compared to the cuprizone mice, which was aligned with the results of LFB and TEM. Gene results showed that PLX treatment reduced CSF1R expression. According to the results, the administration of PLX for 21 days enhanced remyelination by increasing oligodendrocytes in the chronic demyelination model. These positive effects could be related to the reduction of microglia.

17ß-Estradiol Reduces Demyelination in Cuprizone-fed Mice by Promoting M2 Microglia Polarity and Regulating NLRP3 Inflammasome.

Estrogen produces a beneficial role in animal models of multiple sclerosis (MS). The effect of 17ß-estradiol therapy on microglia polarization and neuroinflammation in the corpus callosum of the cuprizone-induced demyelination model has not been elucidated. In this study, mice were given 0.2% cuprizone (CPZ) for 5 weeks to induce demyelination during which they received 50 ng of 17ß-estradiol (EST), injected subcutaneously in the neck region, twice weekly. Data revealed that treatment with 17ß-estradiol therapy (CPZ+EST) improved neurological behavioral deficits, displayed by a significant reduction in escape latencies, in comparison to untreated CPZ mice. Also, administration of 17ß-estradiol caused a decrease in demyelination levels and axonal injury, as demonstrated by staining with Luxol fast blue, immunofluorescence to myelin basic protein, and transmission electron microscopy analysis. In addition, at the transcriptional level in the brain, mice treated with 17ß-estradiol (CPZ+EST) showed a decrease in the levels of M1-assosicted microglia markers (CD86, iNOS and MHC-II) whereas M2-associated genes (Arg-1, CD206 and Trem-2) were increased, compared to CPZ mice. Moreover, administration of 17ß-estradiol resulted in a significant reduction (∼3-fold) in transcript levels of NLRP3 inflammasome and its downstream product IL-18, compared to controls. In summary, this study demonstrated for the first time that exogenous 17ß-estradiol therapy robustly leads to the reduction of M1 phenotype, stimulation of polarized M2 microglia, and repression of NLRP3 inflammasome in the corpus callosum of CPZ demyelination model of MS. The positive effects of 17ß-estradiol on microglia and inflammasome seems to facilitate and accelerate the remyelination process.

Mesenchymal Stem Cells Ameliorate Cuprizone-Induced Demyelination by Targeting Oxidative Stress and Mitochondrial Dysfunction.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. The main causes of MS disease progression, demyelination, and tissue damage are oxidative stress and mitochondrial dysfunction. Hence, the latter are considered as important therapeutic targets. Recent studies have demonstrated that mesenchymal stem cells (MSCs) possess antioxidative properties and are able to target mitochondrial dysfunction. Therefore, we investigated the effect of transplanting Wharton's jelly-derived MSCs in a demyelination mouse model of MS in which mice were fed cuprizone (CPZ) for 12 weeks. CPZ is a copper chelator that impairs the activity of cytochrome oxidase, decreases oxidative phosphorylation, and produces degenerative changes in oligodendrocytes, leading to toxic demyelination similar to those found in MS patients. Results showed that MSCs caused a significant increase in the percentage of myelinated areas and in the number of myelinated fibers in the corpus callosum of the CPZ + MSC group, compared to the CPZ group, as assessed by Luxol fast blue staining and transmission electron microscopy. In addition, transplantation of MSCs significantly increased the number of oligodendrocytes while decreasing astrogliosis and microgliosis in the corpus callosum of the CPZ + MSC group, evaluated by immunofluorescence. Moreover, the mechanism by which MSCs exert these physiological effects was found to be through abolishing the effect of CPZ on oxidative stress markers and mitochondrial dysfunction. Indeed, malondialdehyde significantly decreased while glutathione and superoxide dismutase significantly increased in CPZ + MSC mice group, in comparison witth the CPZ group alone. Furthermore, cell therapy with MSC transplantation increased the expression levels of mitochondrial biogenesis transcripts PGC1α, NRF1, MFN2, and TFAM. In summary, these results demonstrate that MSCs may attenuate MS by promoting an antioxidant response, reducing oxidative stress, and improving mitochondrial homeostasis.

Effect of mesenchymal stem cells on glial cells population in cuprizone induced demyelination model.

Mesenchymal stem cells (MSCs) have a notable potential to modulate immune responses and protect the central nervous system (CNS), mostly by secreting factors that affect inflammation. MSCs have the ability to improve several autoimmune diseases in animal models including multiple sclerosis (MS). MS is a disease of the CNS among adult humans and it is characterized by demyelination, neuroinflammation and gliosis. In this study, we first induced chronic demyelination by cuprizone, followed by intraventricular injection of MSC. Our results showed that MSC significantly decreased microgliosis and astrocytosis by secreting cytokines that have neuroprotective activity including TGF-ß and CX3CL1. Also, downregulation of IL-1ß and TNF-α as inflammatory chemokines was seen along with decreased astrocytes and microglia activation. Finally, these results showed that trophic factors secreted by MSC can increase oligodendrocyte population and remyelination rate by reducing pro-inflammatory factors. These findings demonstrate that MSC could decrease inflammation, gliosis and demyelination with neuroprotective and immunomodulating properties in chronic cuprizone demyelination model. Therefore MSC transplantation can be considered as a suitable approach for enhancing myelination and reducing inflammation in diseases such as MS.

In utero transplantation of neural stem cells ameliorates maternal inflammation-induced prenatal white matter injury.

Prenatal white matter injury is a serious problem due to maternal inflammation leading to postnatal disabilities. In this study, we used the periventricular leukomalacia (PVL) model as a common prenatal white matter injury by maternal administration of lipopolysaccharide (LPS). Neural stem cells (NSCs) have shown therapeutic ability in neurological disorders through a different mechanism such as immunomodulation. Here, we studied the preventive potential of NSCs following in utero transplantation into the embryonic lateral ventricle in an LPS-induced white matter injury model. Pregnant animals were divided into three groups and received phosphate buffered saline, LPS, or LPS + NSCs. The brains of offspring were obtained and evaluated by real-time polymerase chain reaction (PCR), immunohistochemy, enzyme-linked immunosorbent assay (ELISA), terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling (TUNEL), and caspase-3 activity assay. The LPS-induced maternal inflammation degenerated the myelin sheath in the offspring periventricular region which was associated with an increased microglial number, oligodendrocytes degeneration, proinflammatory cytokine secretion, and cell apoptosis. The transplanted NSCs homed into the brain and ameliorated the evaluated parameters. The expression of proinflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α), cell apoptosis and caspase-3 activity were inhibited by NSCs. In addition, Olig2 and myelin basic protein immunohistochemy staining showed that prenatal NSCs transplantation augmented the myelination in the periventricular white matter of offspring. In conclusion, we think that prenatal therapeutic strategies, such as in utero NSCs transplantation, may prevent prenatal white matter injury after birth.

Intrathecal transplantation of Wharton's jelly mesenchymal stem cells suppresses the NLRP1 inflammasome in the rat model of spinal cord injury.

After spinal cord injury (SCI) local inflammation is induced following secretion of interleukin-1beta (IL-1ß) and IL-18. It has been described that the secretion of IL-1ß and IL-18 is mediated by a cytoplasmic multiprotein complex, termed inflammasome. Mesenchymal stem cells (MSCs) have been extensively used for treating inflammatory diseases in which they showed immunomodulation characteristics. We utilized the anti-inflammatory potential of Wharton's jelly mesenchymal stem cells (WJ-MSCs) to target inflammasome complex in rat SCI model. Real time-polymerase chain reaction, western blotting, and ELISA assay were done one week after SCI to measure the expression of the inflammasome components including NLRP1, ASC, and active caspase-1 as well as IL-1ß, IL-18, and tumor necrosis factor-α (TNF-α). The histologic alteration and hind-limb locomotion were evaluated three weeks after injury by nissl staining and Basso, Beattie, Bresnahan (BBB), respectively. Our results showed that WJ-MSCs transplantation significantly decreased the SCI-induced expression of the evaluated factors in both mRNA and protein levels. In addition, WJ-MSCs significantly increased the number of normal-appearance neurons in the ventral horn of spinal cord. Noteworthy, these effects resulted in a significant improvement of motor function recovery. We conclude that inflammasome inhibition may be one of the mechanisms for the anti-inflammatory effect of MSCs in the SCI.

Melatonin preconditioning of bone marrow-derived mesenchymal stem cells promotes their engraftment and improves renal regeneration in a rat model of chronic kidney disease.

Bone marrow-derived mesenchymal stem cells (BMMSCs) transplantation has shown to be effective in treating chronic kidney disease. However, the effectiveness of this strategy is constrained by low homing and survival rate of transplanted cells in the injured organs. Therefore, developing strategies to improve homing and cell survival rate and therapeutic potential in cell-based therapies seems necessary. The purpose of this study is to evaluate the effect of pretreating BMMSCs with melatonin (MT) on the prosurvival and renoprotective of transplanted cells into the irreversible model of unilateral ureteral obstruction. Adult male Sprague-Dawley rats were randomized into four groups: Sham, UUO, UUO + BMMSCs, and UUO + BMMSCs + MT. The results of our study demonstrated that preconditioning with MT enhanced homing of BMMSCs into the injured kidney. MT reduced the number of TUNEL positive transplanted cells in the UUO + BMMSCs + MT group. The UUO + BMMSCs + MT group showed lower expressions of TGF-ß1, α-SMA and TNF-α at both gene and protein levels but higher expression of E-cadherin compared with the UUO + BMMSCs group. In addition, MT preconditioned BMMSCs ameliorated basement membrane disruption and histological status of injured renal tubules and also reduced fibrosis in damaged kidneys. In conclusion, our results show that stem cells pretreated by MT may represent a feasible approach for improving the beneficial effects of stem cell therapy and significantly enhance their survival after transplantation to the injured kidney.

Macrophage polarity in cancer: A review.

Macrophages are the most abundant cells within the tumor stroma displaying noticeable plasticity, which allows them to perform several functions within the tumor microenvironment. Tumor-associated macrophages commonly refer to an alternative M2 phenotype, exhibiting anti-inflammatory and pro-tumoral effects. M2 cells are highly versatile and multi-tasking cells that directly influence multiple steps in tumor development, including cancer cell survival, proliferation, stemness, and invasiveness along with angiogenesis and immunosuppression. M2 cells perform these functions through critical interactions with cells related to tumor progression, including Th2 cells, cancer-associated fibroblasts, cancer cells, regulatory T cells (Tregs), and myeloid-derived suppressor cells. M2 cells also have negative cross-talks with tumor suppressor cells, including cytotoxic T cells and natural killer cells. Programed death-1 (PD-1) is one of the key receptors expressed in M2 cells that, upon interaction with its ligand PD-L1, plays cardinal roles for induction of immune evasion in cancer cells. In addition, M2 cells can neutralize the effects of the pro-inflammatory and anti-tumor M1 phenotype. Classically activated M1 cells express high levels of major histocompatibility complex molecules, and the cells are strong killers of cancer cells. Therefore, orchestrating M2 reprogramming toward an M1 phenotype would offer a promising approach for reversing the fate of tumor and promoting cancer regression. Macrophage switching toward an anti-inflammatory M1 phenotype could be used as an adjuvant with other approaches, including radiotherapy and immune checkpoint blockades, such as anti-PD-L1/PD-1 strategies.

Embryonic intraventricular transplantation of neural stem cells augments inflammation-induced prenatal brain injury.

OBJECTIVE: Prenatal brain injury results from undesirable circumstances during the embryonic development. Current endeavors for treating this complication are basically excluded to postnatal therapeutic approaches. Neural stem cell therapy has shown great promise for treating neurodevelopmental disorders. To our knowledge, this is the first study that investigates the therapeutic effect of in utero transplantation of neural stem cells (NSCs) in inflammation model of prenatal brain injury. METHODS: To induce prenatal injury, time-mated C57BL6J mice were intraperitoneally injected with 50 µg/kg lipopolysaccharide (LPS(on the day 15 of gestation. In the treatment group, NSCs were transplanted into the lateral ventricle of embryos on day 17 of gestation. The expression of GFAP, Iba-1, Olig2, and NeuN were assessed by real time PCR and immunohistochemistry. Changes in IL-6, TNF-α and IL-10 cytokines level, and caspase 3 activity were evaluated in the cortex of pups. RESULTS: Intrauterine transplanted NSCs homed to the brain cortex of offspring. Brain levels of pro-inflammatory cytokines showed a significant downward trend in the NSCs group. Furthermore, NSCs ameliorated inflammation-induced reactive microgliosis and astrogliosis as well as cellular degeneration. Apoptosis inhibition in the treated group was demonstrated by the decline in the caspase 3 activity and dark neurons. CONCLUSION: This study suggests a promising prospect to initiate the treatment of prenatal brain injury before birth by intrauterine transplantation of NSCs.

Therapeutic effect of perinatal exogenous melatonin on behavioral and histopathological changes and antioxidative enzymes in neonate mouse model of cortical malformation.

BACKGROUND: Melatonin, which is an antioxidant and neuroprotective agent, can be an effective treatment for neurological disorders. We assessed the effect of melatonin administration on histological changes, antioxidant enzyme levels, and behavioral changes in a neonate mouse model of cortical malformation. MATERIALS AND METHODS: Cortical malformation was induced by two injections of 15 mg/kg methylazoxymethanol (MAM) on gestational day 15 (E15). Pregnant Balb/c mice were randomly divided into the following six groups: Control (CO), Melatonin (MEL), Luzindole (LUZ), MAM, MEL + MAM1 (co-treatment), and MEL + MAM2 (pretreatment). Melatonin was intraperitoneally injected at a dose of 10 mg/kg daily (from E15 until delivery of from E6 for 20 days after delivery). On postnatal day 31, the activity and anxiety of mice were assessed by open field and elevated plus maze tests, respectively. Histopathological changes in the neonate cortex were studied using hematoxylin and eosin staining and neurofilament immunohistochemistry. Enzyme-linked immunosorbent assays were used to measure the activity of nitric oxide (NO), malondialdehyde (MDA), and antioxidant enzymes, including catalase (CAT), super oxide dismutase (SOD), and glutathione peroxidase (GPX). RESULTS: In the behavioral assessment of neonate mice, a significant increase in the crossing activity and decrease in anxiety were recorded in groups treated with MAM plus melatonin. In histological examination, heterotopic, dysmorphic, and ectopic cells, as well as dyslamination, were seen in the MAM and LUZ groups. However, these defects were attenuated in the MAM plus melatonin groups. Significant reductions were recorded in the SOD and GPX levels in the MAM and LUZ groups compared to the control, while the NO level was increased in these groups. Groups that received MAM plus melatonin showed significant increases in the levels of SOD and GPX and a significant decrease in the level of NO, compared to the MAM group. CONCLUSION: Melatonin increased the crossing activity and decreased the anxiety in the treated mice of the neonate mouse model of cortical malformation. Histologically, the administration of exogenous melatonin in pregnant mice and their neonates had a protective effect on the cerebral cortex of neonates. Also, this effect is elicited by decreasing NO and increasing antioxidative enzymes.

Resveratrol pretreatment enhanced homing of SDF-1α-preconditioned bone marrow-derived mesenchymal stem cells in a rat model of liver cirrhosis.

Stromal cell-derived factor-1α (SDF-1α) has been known to implicate in homing of MSCs, and resveratrol has been reported to have a positive influence on SDF-1 level in the site of injury. In this study, a combined strategy was applied to evaluate bone marrow-derived MSCs (BMSCs) homing to the rat model of liver cirrhosis induced by common bile duct ligation (CBDL): (1) pretreatment delivery of resveratrol into the cirrhotic liver, and (2) transplantation of ex vivo BMSC preconditioning with SDF-1α. BMSCs were preconditioned with 10 ng/µL SDF-1α for 1 h and then labeled with the CM-Dil. Cirrhosis was induced by CBDL. Animals received intraperitoneal injection of resveratrol for 7 days, started on day 28 of CBDL post-operative. On day 36 post-operative, 1 × 106 of SDF-1α-preconditioned BMSCs was injected via caudal vein. Animals were sacrificed at 72 h post-cell transplantation. Immunofluorescence and flow cytometry assessments showed that the BMSC+SDF+RV group had an increased rate of homing into the liver, but it had a decreased rate of homing into the lung and spleen, as compared with the other groups (P < 0.05). The BMSC+SDF+RV group showed high protein expression of SIRT1, but low protein expression of p53 in the liver (P < 0.05 vs other groups). CXCR4 and matrix metalloproteinase (MMP)-9 highly expressed in SDF-1α-preconditioned BMSCs in vitro, and that AKTs and CXCL12 expressed in injured liver undergoing resveratrol injection. Our findings suggest that reseveratrol pretreatment prior to SDF-1α preconditioning could be a promising strategy for designing cell-based therapies for liver cirrhosis.

Inductive role of sustentacular cells (Sertoli cells) conditioned medium on bone marrow derived mesenchymal stem cells / Papel inductivo del medio acondicionado de células sustentaculares (células de Sertoli) en las células madre mesenquimales derivadas de la médula ósea

RESUMEN: Las células madre de la línea germinal masculina son factores clave para la espermatogénesis masculina y la fertilidad. Las células sustentaculares (células de Sertoli) como células somáticas juegan un papel fundamental en la creación de un microambiente esencial para la auto-renovación y diferenciación de las células de la línea germinal masculina. Las células madre mesenquimales son reconocidas como células auto-renovables y multipotentes capaces de diferenciarse en múltiples tipos de células. La generación de células germinales masculinas a partir de células madre mesenquimales puede proporcionar un método terapéutico para tratar la infertilidad masculina. En este estudio, las células mesenquimales derivadas de la médula ósea (BMMSCs) se recuperaron de la médula ósea de ratones de 6-8 semanas de edad del Instituto de Investigación Médico Naval (NMRI). En el estudio se aislaron las células sustentaculares y se enrriquecieron usando placas revestidas con lectina. Se obtuvo el medio de condición celular después de diferentes intervalos de tiempo. Posteriormente se cultivaron las BMMSC con diferentes concentraciones de SCCM y medio de Eagle modificado por Dulbecco (DMEM) en diversos momentos. Se evaluaron marcadores específicos de células de línea germinal usando la reacción en cadena de polimerasa transcriptasa inversa (RT-PCR) e inmunocitoquímica. Los resultados mostraron que las BMMSCs cultivadas con SCCM durante 48h exhibieron transcritos específicos de línea germinal (Mvh, Iid4, piwil2) (p <0,05) y marcadores (Mvh, Scp3). Nuestros resultados indican que el cultivo de BMMSCs con SCCM puede conducir a la diferenciación efectiva de BMMSCs en células germinales y proporcionar una estrategia de tratamiento para la infertilidad masculina.

SUMMARY: Male germ line stem cells are key factors for male spermatogenesis and fertility. Sustentacular cells (Sertoli cells) as somatic cells play a pivotal role in creating essential microenvironment for the self-renewal and differentiation of the male germ line cells. Mesenchymal stem cells are recognized as self-renewing and multipotent cells able to differentiate into multiple cell types. The generation of male germ cells from mesenchymal stem cells may provide a therapeutic method to treat male infertility. In this study, Bone marrow derived mesenchymal cells (BMMSCs) were retrieved from the bone marrow of 6-8-week old Naval Medical Research Institute (NMRI) mice. Sustentacular cells (Sertoli cells) were isolated and made rich using lectin coated plates. Sustentacular cell condition medium (SCCM) was collected after different time intervals. Then the BMMSCs were cultured with different concentration of SCCM and Dulbecco's Modified Eagle's medium (DMEM) at various times. Specific markers of Germ line cells were evaluated by using Reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry. The results showed that BMMSCs cultured with SCCM for 48h exhibited germ line specific transcripts (Mvh, Iid4, piwil2) (p< 0.05) and markers (Mvh, Scp3). Our findings represent that culturing BMMSCs with SCCM may lead to effective differentiation of BMMSCs into germline cells and provide a treatment strategy for male infertility.

Progesterone therapy induces an M1 to M2 switch in microglia phenotype and suppresses NLRP3 inflammasome in a cuprizone-induced demyelination mouse model.

Demyelination of the central nervous system (CNS) has been associated to reactive microglia in neurodegenerative disorders, such as multiple sclerosis (MS). The M1 microglia phenotype plays a pro-inflammatory role while M2 is involved in anti-inflammatory processes in the brain. In this study, CPZ-induced demyelination mouse model was used to investigate the effect of progesterone (PRO) therapy on microglia activation and neuro-inflammation. Results showed that progesterone therapy (CPZ+PRO) decreased neurological behavioral deficits, as demonstrated by significantly decreased escape latencies, in comparison to CPZ mice. In addition, CPZ+PRO caused a significant reduction in the mRNA expression levels of M1-markers (iNOS, CD86, MHC-II and TNF-α) in the corpus callosum region, whereas the expression of M2-markers (Trem-2, CD206, Arg-1 and TGF-ß) was significantly increased, in comparison to CPZ mice. Moreover, CPZ+PRO resulted in a significant decrease in the number of iNOS+ and Iba-1+/iNOS+ cells (M1), whereas TREM-2+ and Iba-1+/TREM-2+ cells (M2) significantly increased, in comparison to CPZ group. Furthermore, CPZ+PRO caused a significant decrease in mRNA and protein expression levels of NLRP3 and IL-18 (~2-fold), in comparison to the CPZ group. Finally, CPZ+PRO therapy was accompanied with reduced levels of demyelination, compared to CPZ, as confirmed by immunofluorescence to myelin basic protein (MBP) and Luxol Fast Blue (LFB) staining, as well as transmission electron microscopy (TEM) analysis. In summary, we reported for the first time that PRO therapy causes polarization of M2 microglia, attenuation of M1 phenotype, and suppression of NLRP3 inflammasome in a CPZ-induced demyelination model of MS.

Evaluation of the Effect of Pentoxifylline on Cisplatin-Induced Testicular Toxicity in Rats.

Chemotherapy is associated with male infertility. Cisplatin (cis-diamminedichloro-platinum (II) (CDDP) as a chemotherapy medication used to treat a number of cancers has been reported to most likely induce testicular toxicity. Administration of antioxidants, such as pentoxifylline (PTX) may reduce some Adverse Drug Reactions (ADRs) of CDDP. Therefore, this study investigated the potentially protective effects of PTX on CDDP-induced testicular toxicity in adult male rats. For this purpose, 42 male rats were randomly divided into 7 groups. The rats were orally pretreated with PTX at the 3 doses of 75, 150, and 300 mg/kg once a day for 14 successive days. On the 14th day of the study, they were intraperitoneally (IP) administered with a single dose of CDDP (7 mg/kg). Finally, the sperm/testis parameters, serum levels of reproductive hormones, including testosterone, Luteinizing Hormone (LH), and Follicle Stimulating Hormone (FSH) as the pivotal endocrine factors controlling testicular functions, and histopathological changes of testis tissue were examined. Pretreatment with the two doses of 75 and 150 mg/kg PTX indicated significant increases in the sperm count and motility induced by CDDP administration. The right and significantly left testis weights were decreased following the treatment with 300 mg/kg of PTX plus CDDP. However, 75 mg/kg of PTX plus CDDP showed the best near-to-normal histopathological features. The results demonstrated that PTX alone enhanced some parameters, such as the sperm count, while reducing other parameters, including sperm fast motility and germ layer thickness. Furthermore, despite testosterone or LH levels, the mean serum FSH level was significantly augmented by the doses of 75 and 150 mg/kg. It was concluded that PTX administration cannot reduce CDDP-induced testicular toxicity even at high doses (e.g., 300 mg/kg), while it seemed to partially intensify CDDP toxicity effects at a dose of 75 mg/kg. Thus, further research is required in this regard.

Preconditioning with melatonin improves therapeutic outcomes of bone marrow-derived mesenchymal stem cells in targeting liver fibrosis induced by CCl4.

Preconditioning of mesenchymal stem cells (MSCs) with melatonin (MT) has shown promising results in animal models of myocardial infarction, renal ischemia and cerebral ischemia. Here, we use this strategy in the liver fibrosis induced by CCl4. There were five groups: normal, CCl4, CCl4 + vehicle, CCl4 + BMMSCs and CCl4 + MT-bone marrow (BM)-derived MSCs (MT-BMMSCs). CCl4 was injected twice weekly for 8 weeks and treatment either with cells or vehicle was performed at the beginning of week 5 with a single dose. BMMSCs were preconditioned with MT for 24 h before injection. MT-BMMSCs had a high ability of homing into the injured liver (P ≤ 0.05 vs. BMMSCs). The CCl4 + MT-BMMSCs group showed higher percentage of glycogen storage but lower percentage of collagen and lipid accumulation (P ≤ 0.05 vs. CCl4 + BMMSCs). The CCl4 + MT-BMMSCs group showed lower expressions of transforming growth factor-ß1 (TGF-ß1) and Bax and lower content of sera alanine aminotransferase (ALT) but higher expressions of matrix metalloproteinases (MMPs) and Bcl2 compared with the BMMSCs group (P ≤ 0.05). The results showed the better therapeutic outcomes of MT preconditioning by probably improving cell homing and also better maintenance of the balance between matrix degrading and accumulating factors.