Anatomical Sciences Journal
Year:2007 | Volume:5 | Issue:19-20|Papers Count:12

Purpose: Isolation, induction of neural and glial differentiation and evaluating the expression of Nucleostemin, ZFX, Hoxb-4, Sox-9 & Bmi-1 self renewal genes in adult mouse neural stem cells Materials and Methods: Breifly, for isolation of neural stem cells, frontal part of adult mouse brain was minced in PBS and digested by enzyme solution, containing hyaloronidase and trypsin. Isolated cells were cultured in medium supplemented by EGF, bFGF. After one week, primary neurospheres were appeared in culture medium which differentiated into neural and neuroglial cells, after transferring to poly-L-ornyethin coated dishes. Differentiated cells were examined by morphological, immunocytochemical and molecular evaluations.Results: Our results showed that isolated cells from periventricular area of mouse adult brain can be expanded in medium containing EGF, bFGF and Fetal calf serum and differentiated into neural and neuroglial cells. Immunocytochemical studies for GFAP, MAP2 and O4 genes, proved the differentiation. Our results also showed that Nucleostemin, ZFX, Hoxb-4, Sox-9 & Bmi-1 are expressed in mouse neural stem cells.Conclusion: Mouse adult brain contains a type of stem cell which can be differentiated into neural and neuroglial cells. In addition, expression of Sox-9, Nucleostemin and Bmi-1 self renewal genes in undifferentiated neural stem cells can be considered as a proof for stem cell identity of isolated cells. This study is the first report which shows the expression of ZFX and Hoxb-4 genes in neural stem cells.

Purpose: The purpose of this study was the investigation of thermal and mechanical Allodynia after BMSCs grafting in the Spinal Cord Contusion of rat Materials and Methods: In this study used 40 female Sprague- Dawley 6-8 week old that 33 rats received vertebral laminectomy to expose spinal cord (L1 vertebral level). The cord was then contused with the weight drop device. Experimental groups consist group 1 (Intact) rats without intervention (n=7); group 2 and 3: 16 rats given PBS alone without cell administration respectively 10 rats intravenously (vehicle1) and 6 rats intraspinally (vehicle2); group 4 and 5: 17 rats given BMSCs after 4 passages respectively 10 rats intravenously and 7 rats intraspinally. Grafts were done 1 week after injury. Behavioral tests were done for 10 weeks (after injury). For thermal testing, rats were placed on a hotplate. Mechanical sensitivity was assessed with Von Frey Hairs. Tracing of the cells at the injured site was performed immunohistochemically and then astrocyte differentiation was detected with the double-Staining method. Results: BMSCs-engrafted rats showed significant decrease in withdrawal threshold and time latency to licking of hindpaws in comparison with vehicle groups. Also there was astrocyte differentiation.Conclusion: The results demonstrate that intraveous and intraspinal grafts of BMSCs in the cases of spinal cord contusion enhance thermal and mechanical Allodynia. Also Allodynia could be positively correlated with the number of astrocytes.

Purpose: The motor control deficits exhibited with Parkinson’s disease (PD) are particularly well documented for sequencing tasks. The purpose of this study was to evaluate the neuroprotective potential of trans-resveratrol administration in an experimental model of PD.Materials and Methods: 1 week before (baseline) the surgery rats were tested with systemic apomorphine (0.5 mg/kg i.p.) to evaluate the behavioral effect of the nigrostriatal lesion (turning behavior). The number of full rotations performed by the animals was registered at 10-min intervals for 60 min starting 1 min after apomorphine injection. The same procedure was repeated after 14 days post lesioning by 6-OHDA in the left striatum. Rotational response was expressed as the number of net full (360°) turns per hour. The number of contralateral rotations was counted as positive scores for apomorphine. Net number of rotations was defined as the positive scores minus the negative scores.Results: There were no significant differences among the groups at baseline (before surgery). Statistical analysis of the total net number of rotations made over a 60-min period 2 weeks after the surgery showed that apomorphine caused a very significant contralateral turning in the rats of the L group (P<0.001) and induced less significant rotations in the L+RSV group (P<0.05) in comparison with the SH group. Moreover, the group L+RSV showed a significant reduction of rotations (P<0.001) when compared with the L group.Conclusion: In conclusion, administration of trans-resveratrol effectively counteracted progressive degeneration of nigrostriatal neurons caused by intrastriatal injection of 6-OHDA, thus lending further support to the neuroprotective potential of the drug.

Purpose: The aim of present study was to study the effects of low-level helium–neon laser irradiation on biomechanical properties of tibiae partial bone defect in streptozotocin–diabetic rat.Materials and Methods: Twenty male rats were used .Diabetes was induced in rats by one time intraperitoneal injection of 60 mg/kg streptozotocin .After one month ,under sterile conditions and general anesthesia one partial bone defect were made in mid portion of right tibia of rats .Rats were divided into control and no 1 and no 2 experimental groups . Rats of no 1 and 2 experimental groups were received he-ne laser with 2.1 J/cm² and 11.6 J/cm² energy densities three times a week for six weeks. After that rats were killed and their tibiae were extracted and were examined by three point bending biomechanical test. Data were analysed by ANOVA and expressed as mean±SEM.Results: The energy absorption (N/mm) of no1 experimental group. Control and no2 experimental groups were 52±45, 44.2±35.2 and 36.3±20.1 respectively. Load at break (Nmm) of no1 and no2 experimental group and control group were 34.2±13.2, 32.6±18.6 and 31.8±27.4 respectively .Maximum force per square mm of no1 experimental group, control and no2 experimental group were 8.2±3.57, 8.2±4.4 and 7±4.96 respectively .There were not significant differences between all above mentioned parameters and also elastic stiffness, maximum force and bone surface area of groups.Conclusion: He-Ne laser irradiation on tibias of partial bone defect in streptozotocin diabetic rats did not cause positive effects on fracture healing from biomechanical evaluation point of view.

Purpose: Despite remarkable progresses have been achieved in the field of somatic cell nuclear transfer (SCNT), there is little information regarding the effect of donor cell type on the efficiency mammalian somatic cell cloning in vitro. This study compared in vitro developmental competency of sheep enucleated oocytes reconstructed with either fibroblast or cumulus cells.Material and methods: Adult fibroblast cells were taken from skin biopsies of one male and one female adult sheep. Cumulus cells were harvested from the same female sheep. Direct whole cell injection procedure was used for embryo reconstitution. Activated reconstructs were cultured in TCM+10%FCS medium up to days 7 post activation whereas, in vitro fertilized oocytes served as control. Results: Although the rate of day 7 blastocyst formation in cumulus donor cells (19.7%) were greater than the male and female fibroblast donor cells (17.5% and 15.7% respectively), these differences were not significant. Control group, on the other hand, induced significantly greater blastocyst rate (29.7%) rather than all the three treatment groups.Conclusion: Both of cumulus and fibroblast can be used as donor cell for ovine somatic cell nuclear transfer. Since no significant difference was observed in term of blastocyst rates between the three SCNT groups, therefore concluding donor cell type has no significant effect on the overall efficiency of in vitro production of cloned sheep blastocyst.

Purpose: The purpose of this study was to develop an appropriate medium for in vitro maturation (IVM) of immature mouse oocytes.Materials and Methods: Germinal vesicle of female NMRI mouse oocytes (6-8 weeks old) were collected from ovaries and cultured in maturation medium MEM-a, supplemented with: 100 mlIU/ml rFSH+7.5 IU/ml hCG+5% FCS (Control group) and 2mM all- trans retinoic acid (t-RA) in presence or absent of granulose cells. Ethanol (Sham group) 0.2% (v/v) used as solvent. After 24 hours the matured oocytes were fertilized with spermatozoa in T6 medium and cultured for 5 days. Cultured immature oocytes development to the morula and blastocyst stages was studied.Results: The retinoic acid supported progression and resumption of meiosis and also increased advancing the oocytes to Metaphase II, formation of morula and blastocyst compared to control group. When there were not 2mM t-RA and granulose cells in IVM medium, significantly lower maturation rates were observed, followed by a decrease in the percentage of embryos reaching the blastocyst stage. Whereas, when 2mM t- RA and granulose cells monolayer were present in the IVM medium, better results in comparison with control group were obtained.Conclusion: Results attest that co-culture of granulose cells with 2mM all-trance retinoic acid during in vitro maturation enhanced mouse oocytes maturation and improved embryonic development.

Purpose: Among antiepileptic drugs, valproic acid (VA) is a well known teratogenic agent. We previously reported that maternal (VA) administration of VA during critical periods of rat pregnancy can produce very rare CNS defect, syringomyelia. The purpose of the present study was whether VA can affect other organs, based on this finding, we studied the teratogenic effects of it on rat developing skeletal system.Materials and Methods: Based on this experimental study 3 experimental groups of pregnant wistar rats were received single dose of 800 mg/kg VA on gestational days 9 -11 respectively. 3 Control groups were received the same volume of physiological serum at the same periods. On day 18th of gestation all pregnant rats were anesthetized and their fetuses were examined and, histochemical study around their vertebral region sections Carried out.Results: Our finding indicated different kinds of malformations in experimental groups which was consist of growth retardation, embryonic absorbtion as well as skeletal system defect by special regard in vertebral column. These data revealed that the rate of spina bifida and vertebral abnormal flexions in experimental group 1 increased significantly in Comparison experimental groups 2 and 3.Conclusion: These findings suggest that, day 9th of rat pregnancy is critical period for vertebral column development and during this period of time VA or its metabolites may be interfere with developmental phenomena which can produce vertebral defects such as spina bifida.

Purpose: The present study was designed to investigate the effect of different doses of Staurosporine on differentiation of PC-12 cell line into differentiated neuronal cells. Materials and Methods: PC-12 cell line as a useful model system for neurobiological studies was maintained in RPMI 1640 supplemented with 10% FBS. Staurosporine were added to culture medium in different concentrations (110, 214, 316 and 1000nM), then morphology and apoptotic index were assessed by total neurite length estimation and TUNEL staining.Results: Data showed that total neurite length of PC-12 cells that were treated with 110, 214, 316 and 1000nM was 44±9.3, 153±15.0, 104±10.4 and 31±9.6μm respectively. Therefore PC-12 cells that were treated with 214nM Staurosporine had the greatest neurite length in compression with the other concentrations (P<0.05). In addition apoptotic index in these concentrations was 3±0.7,4±0.8,8±1.0 and 10±1.1 respectively, Therefore PC-12 cells that were treated with 110 and 214nM Staurosporine had minimal apoptotic index (P<0.05).Conclusion: Finally it can be concluded that 214nM concentration of Staurosporine is an optimum concentration for differentiation of neuronal cell lines to differentiated neuronal cells.

There are two main categories for stem cells a cording to their origin: Embryonic Stem Cells and Adult Stem Cell. Mesenchymal stem cell, supporting hematopoetic stem cells in bone marrow, can regenerate tissues such as bone, cartilage, muscle, tendon and fatty tissue. These cells were recognized for the first time by Friedenstein and Petrokova who could isolate theme from rat bone marrow.Mesenchymal Stem Cells can be obtained easily from bone marrow although they can be isolated from other tissues such as fetal blood, cord blood and amnion fluid and membrane. The frequency of these cells in bone marrow is 0.001-0.1 % of cell population. Aspiration of bone marrow from iliac crest, femur and tibia contains of mesenchymal stem cells which can be easily expanded In Vitro. These cells isolated from bone marrow according to their ability to adhere culture area and can be expanded according to the standard protocols. There is still no known marker(s) for mesenchymal stem cells; however, recent studies showing some specific marker for these cells. They lack hematopoetic stem cells marker such as CD14, CD45, CD34; endothelial markers such as CD34, CD31 and von-Willberand (VWF) factor. Mesenchymal stem cells express a few of adherent molecules such as CD44, CD29, CD90 and stromal cell markers SH-2, SH-3, SH-4 and also receptors like IL-1 and TNF receptors. These kinds of cells can be transdifferentiated to cells of three lineages such as neuron, hepatocyte and cardiomyocyte like cells. The accessible sources and easily expansion of these cells and also being autologe make them ideal for therapeutic usage, but low frequency of these cells in the bone marrow and need of additional componenet such as serum (especially animal based serum) for good expansion in the culture media is the main problem in this field, however researchers are trying to expand mesenchymsl stem cell in serum free medium.In this article we will first talk about the biology of mesenchymal stem cell, their Immunologic properties, their application in regenerative medicine and clinical application and at the end we will talk about isolation and expansion of these cells in a protocol manner.