[Survival and migration of bone marrow derived neurosphere after transplantation into subretinal space in albino rat AMD model]

Background and Aim: degenerative retinal diseases are among the common causes of blindness in the world. The purpose of this study was to investigate transplantation of neurosphere derived from bone marrow tissue into subretinal space in age related macular degeneration induced by injection of sodium iodate in animal model

Materials and Methods: 40 mg/kg of sodium iodate was injected into retro-orbital sinus of albino rats. Then histological investigation by flat-mount and hematoxylin and eosin staining was performed after 30 days. Bone marrow stromal stem cells isolated from albino rats femur, were cultured in the differentiation medium and induced into floating neurosphere. Differentiated cells were labeled with nuclear anti-BrdU and were transplanted into subretinal space. Seven days after injection, sections were prepared, and survival, migration and also arrangement of transplanted cells were investigated by immunohistochemistry

Results: three days after sodium iodate injection, the pathological changes such as increased autofluorescence, hypertrophy and multinuclearity in retinal pigmented epithelium were observed. Histological investigation showed disorganization of outer segment of photoreceptors and also changes in the retinal pigmented epithelium. Immunohistochemsitry findings, seven days after injection, showed that transplanted cells survived in subretinal space and could migrate into both retinal pigmented epithelium and the retinal layer and finally integrated with host tissue

Conclusion: due to accessibility, mesenchymal stem cells are regarded as a good source for transplantation. Potential of differentiation to neural linage and also survival ability and migration of these cells after transplantation could be regarded as a new way for the treatment of retinal degenerative diseases

[An in vitro study of the effect of sodium selenite on telomere length and telomerase activity of bone marrow stromal cells in aged rats]

Background and Aim: the number and potential of proliferation and differentiation of bone marrow mesenchymal stromal cells decreases with age. These changes reduce efficacy of autologous transplantation in old people. The purpose of this study was to evaluate the effect of sodium selenite on telomere length and telomerase activity of bone marrow stromal cells [BMSCs] in aged rats

Material and Methods: the BMSCs collected from aged male rats were cultured and treated with different concentrations of sodium selenite for 72 h. We evaluated the effect of sodium selenite on the proliferation potential of these cells using trypan blue exclusion test. Then, we evaluated the efficacy of the effective concentration of sodium selenite on telomerase activity, telomere length and the related telomerase gene expression. Telomerase activity was assessed byPCR-ELSA method and telomere length, and its related gene expression was assessed by the real time PCR technique

Results: use of sodium selenite at the concentration of 100nM led to significant increase in the proliferation of BMSCs and decreased telomere length in the aged rats compared to the control group, although the difference was not significant. Telomerase activity and the related telomerase gene expression did not show any change

Conclusion: sodium selenite improved proliferation of BMSCs of the aged rats

[Effect of hydro-ethanolic extract of chamaemelum nobile on cell prolifration and apoptosis of rat hipocample neural stem cells in the oxitative stress condition]

Neural stem cells can difrentiate to mature neural cells. Neural stem cells can migrate and repair the damage neural tissue. This study was done to determine the effect of hydro-ethanolic extract of Chamaemelum nobile on cell prolifration and apoptosis of rat hipocample neural stem cells in the oxitative stress condition. In this experimental study, neural stem cells were isolated from hippocampus of neonatal rat brain. Isolated neural stem cells were treated at 200, 400, 600, 800 and 1000 microg/ml of hydro-ethanolic extract of Chamaemelum nobile for 48h. Cells proliferation rate were evaluated by MTT assay. Anti-apoptotic property of hydro-ethanolic extract of Chamaemelum nobile evaluated using TUNEL assay method. Proliferation of neural stem cells were significantly increased in Chamaemelum nobile extract group in comparision with control [P<0.05]. The rate of apoptotic cells were significantly reduced in Chamaemelum nobile extract group compared to control [P<0.05]. The hydrethanolic extract of Chamaemelum nobile increases proliferation rate and reduces apoptosis of neural stem cells in the oxitative stress condition

Time-dependent changes of immunologic responses after burn injury and immunomodulation by cimetidine and pyrimethamine in an animal model

Severe suppression of the immune system is the major cause of infections following burn injury. The aim of this study was to investigate the time-related alterations of immune responses following thermal injury in an animal model and also to modulate immune responses by use of the immunomdulators cimetidine and pyrimethamine. Male Balb/c mice were anesthetized and given a 10% total body surface area full-thickness burn. The timedependent changes of delayed type hypersensitivity [DTH] and antibody responses to sheep red blood cell [SRBC] were assessed at post-burn days [PBD]. The effects of different doses of cimetidine and pyrimethamine on DTH response were also quantitated at 10 PBD. Marked suppression of DTH response occurred during 30 days after burn trauma, with maximal suppression occurring between 10 to 14 days after burn injury. Simultaneously the antibody response to SRBC was significantly increased after thermal trauma. Cimetidine [at doses of 10 and 15 mg/kg] and pyrimethamine [at doses of 5 and 10 mg/kg] significantly augmented DTH response after thermal injury. These results showed that the severe time-dependent alterations occurred in DTH and antibody responses following burn injury. Cimetidine and pyrimethamine also restore burn-induced suppression of DTH response following thermal trauma

Study of neuroprotective effects of green tea antioxidant on spinal cord injury of rat

Recent studies revealed the neuroprotective effects of green tea antioxidant on experimental cerebral ischemia, but these effects on spinal cord injury [SCI] has not yet been studied. Rats were randomly divided into three groups of 18 rats each as follows: sham group [laminectomy], control group [SCI] and experimental group [EGCG]. Spinal cord samples were taken 24 h after injury and studied for determination of lipid peroxidation levels and TUNEL reaction. Behavioral testing was performed weekly upto six weeks post-injury. Then, the rats were euthanized for histopathological assessment. The results showed that lipid peroxidation levels were significantly decreased in experimental group. EGCG significantly reduced TUNEL-positive rate. Also, EGCG reduced significantly lesion area, and improved behavioral function more than the control group. EGCG treatment decreased secondary spinal cord injury

study on in-vitro transdifferentiation of rat bone marrow stromal cells into neuroepithelial-like cells

Bone marrow stem cells [BMSCs] are a rich source of stem cells and may represent a valid alternative to neural or embryonic stem cells by replacing the autologous damaged tissues in neurodegenerative diseases. In this study, we attempted to devise a protocol for the induction of BMSCs into neuroepithelial-like cells [NELCs]. Rat BMSCs were isolated from the long bones of adult Sprague-Dawley rats. Their purity in the 4th passage was evaluated with fibronectin by immunocytochemistry, and the stemness marker Oct-4 was assessed by RT-PCR technique. The cells were expanded and induced in the induction stage. The BMSCs were incubated with either beta-mercaptoethanol [micro ME] [1 mM], dimethyl sulfoxide [DMSO] [2%] or biotylated hydroxyanisol or butylated hydroxyanisol [BHA] [200 micro M] in beta-MEM medium without fetal bovine serum [FBS]. They were washed with phosphate buffer saline [PBS] and proceeded to the 2nd phase of induction, where the induction medium was changed with beta-MEM and 15% FBS containing all-trans retinoic acid [RA] [1 micro M] [for 3 days]. Then, the expression of the markers was assessed with GFAP, nestin and neurofilament 68 antibodies, respectively and the expression of Oct-4 and NeuroD was evaluated by RT-PCR. The purity of the BMSCs at the 4th passage was more than 92%. The mRNA of Oct-4 was expressed in these cells. Induction of BMSCs by DMSO-RA could differentiate NELCs significantly more than beta ME-RA and BHA-RA. The transdifferentiation of NELCs was evaluated by nestin antibody and NeuroD mRNA expression; later markers expressed very low detectable level in BMSCs. But the differentiation of BMSCs into astrocytes was less in all of the experiment groups that is estimated GFAP antibody. The application of DMSO-RA can transdifferentiate BMSCs into NELCs in- vitro

[ Study on Transdifferentiation of Bone Marrow Stromal Cells into Neuronal and Glial-Like Cells In Vitro by Different Inducers]

There are some evidences to suggest that bone marrow stromal cells [BMSCs] not only differentiate into mesodermal cells, but also adopt the fate of endodermal and ectodermal cell types. BMSCs can be a valuable cell source as an autograft for clinical application involving regeneration of the central nervous system. Bone marrow stromal cells can he expanded rapidly in vitro and can he differentiat into neuronal- and glial-like cells. In this study, we attempted to devise a protocol or protocals for the induction of BMSCS into neuroepithelial- and neuroglial-like cells. Bone marrow was extracted from the femur and tibia of adult rat, and then bone marrow stromal cells with 4 passages were proliferated and cultured and then were evaluated with fibronectin by immunocytochemistry and Oct-4 by semi quantitative RT-PCR techniqucs. Also in this stage expression of Nestin. NF68, GFAP and 04 antibodies respectively markers of neuroepithelia1, neuron astrocytes and oligodendrocytes cells, were assessed. Rat BMSCs were differentiatec; by two consequent indductors into neuroepithelial. neuronal and glial-like cells. At pre-induction stage dimethyl sulfoxide [DMSO], beta-mercaptoethanol [[3ME] or biotylated hydroxyanisol [BHA] were separattly and without fetal bovine serum [betaBS] addled to alpha minimal essential medium [alfa-MEM], and then a induction stage medium was replaced by retinoic acid [RA] and 15% FBS in alfa-MEM. Four days later, expressions of neuronal and glial markers were assessed. In addition, expression of NeuroD and Oct-4 mRNA were assessed in these cells. More than 92% of BMSCs was fibronectin positive at passage 4. A few percent of BMSCs differentiated into neuroepithelial and neuron-like cells but no astrocyte and oligoclendiocyte-like cell were detected. Oct-4 mRNA was highly expressed in these cells while NeuroD mRNA expression was not detected Induction of BMSCs by DMSO-RA differentiated BMSCs into neuroepithelial and neuronal-like cell significantly compare to betaME-RA and BHA-RA. Transdifferentiation of the treated BMSCs into astrocytes and oligodendrocyte-like cells was less than 5%. Indluction of BMSCs by DMSO-RA resulted in expression of NeuroD niRNA but Oct-4 mRNA was not expressed in none of treatment groups. Induction of BMSCs by different inducers specially DMSO-RA could highly transdifferentiate BMSCs into neuroepithelial and neuronal-like cells, whereas glial-like cells transdifiŠrentiation was very low

effects of cold injury on sensorimotor cortex of mouse brain

Cold injury has been used as a useful model for studies of traumatic brain injury. This model is used to induce brain edema. Brain edema is a pathophysiological condition of increased brain water content due to a variety of coexisting brain injuries, including ischemia, trauma, tumor and infection. The purpose of this study was to evaluate the effects of cold injury on sensorimotor cortex of mouse. 15 male NMRI mice, 6-8 weeks old every one of them with the weight of 30-35gr [5 mice per group] were studied. To produce cold injury a metal probe cooled with liquid nitrogen and was applied to the surface of the intact skull above the parietal lobe by force of 100 gr for 30 sec. Brains were removed 72h after cold injury, 10 micro m serial sections were obtained following fixation, processing and blocking of the brain. The sections were stained using creysl fast violet. Sensorimotor cortex was recognized and 35 fields were chosen randomly to be studied. To do morphometrical study on cortex of frontal and parietal lobes, the cells with a nucleus diameter of 10 micro m were determined. The data were analyzed by means of ANOVA and TUKEY'S HSD test. The results indicated that the number of alive neurons in the model group was significantly [p<0.05] lower than that of control groups. Cold injury decreases the number of normal cells of sensory motor cortex and leads to cell death

Antiapoptotic effect of deprenyl in sensory neurons of rat dorsal root ganglion after sciatic nerve transection

Study on antiapoptotic effect of Deprenyl insensory neurons of dorsal root ganglion of rat. White Sprague-Dawley rats. The regions ofoperation were sciatic nerve in midthigh and L dorsal rootganglion. New born rats [3rd days] were divided intofive groups and their left side sciatic nerves weretransected at midthigh region.Five test groups wereadministered by deprenyl in different doses of: 0.1, 1, 10,25 and50 mg/Kg and the antiapoptotic effect of drug werecompared with control group.Statistical analysis: The values obtained from each testgroups were compared with control group by t-test andoneway ANOVA. The results showed that treatment of animal withdeprenyl prevented reduction in neurons number, in dosedependent manner. The group treated with dose 25mg/Kgshowed the better results. Deprenyl is able to prevent the induction ofapoptosis in axotomized and nonaxotomizedneurons

Sex determination of human fetus using nested-PCR

One of the goals of modern genetics is to develope safe prenatal diagnostic tests which do not constitute any risk to the fetus. One potentially non-invasive approach for doing so is to obtain fetal material from the maternal circulation. In this study we obtained maternal blood from the pregnant women and tried to determine sex of fetus by nested-PCR. To determine the sensitivity of the PCR methods, artificial samples were prepared first by mixing whole blood of adult males with that of the adult females. After performing DNA extraction, PCR was optimized on these samples. Whole blood samples were then obtained from 70 pregnant women in 8 to 12 weeks of gestation after considering the medical ethics. In this study we tried to extract the DNA of the white blood cells [WBC] as well as free DNA of serum and plasma of the maternal bloods and performed Nested-PCR using primers flanking the specific-sequence of Y-chromosome or Sex determining region on Y [SRY]. If the fetus was male, the size of amplified fragment of the first round product was about 470 base pairs and second round was 254 base pairs. After delivery, Only 32 out of 70 of the samples could be followed and results showed that 18 were male. However, the results of the nested-PCR examination of the serums indicated that 21 of the fetas were male and examination of the WBCs showed that 22 were male, in each group 3 and 4 cases were misinterpreted respectively. So the accuracy of sex determination of the samples in serums and WBCs were 90.62% and 81.25% respectively which is almost similar to the recent reports [91.5%]. We could not obtain good results from the free DNA of the plasma. We have obtained relatively positive results from the analysis of maternal blood and if we could follow all cases to see the results of all deliveries, we could then reconfirm the results of our research. Anyway we hope this project could be able to introduce this new approach as means of noninvasive prenatal diagnosis for detection of common inherited diseases in Iran

[Differential expression of two different variants of survivin during mouse brain development]

Introduction: Survivin, an inhibitor of apoptosis [IAP] containing one baculoviral IAP repeat [BIR] domain, has been reported to be able to regulate both cellular proliferation and apoptotic cell death. In the present study, we evaluated the differential expression of different variants of survivin during prenatal and postnatal development of brain in mice

Material and Methods A total of 27 NMRI mice were categorized into 9 age groups and brain specimens were obtained accordingly [n=3 per each group]: 11 and 17 days embryos and newborn from which the remaining brains were collected by intervals of 5 days up to 1 month of age. Total RNA was extracted from each brain and Reverse Transcription was performed by oligo dT and M-MLV enzyme. cDNAs were amplified with primers specific for survivin and beta2 microglobulin [as an internal control] via polymerase chain reaction technique

Results: We demonstrated that survivin is expressed during both fetal and postnatal development of brain in mice. RT-PCR performed on survivin showed two different variants of survivin with different intensities. The expression of the bigger variant [survivin140] during both prenatal development and at birth was significantly higher than its postnatal one

Conclusion: Our data suggests that the expression of survivin140 in brain is developmentally regulated; such a regulation may play a role in homeostasis of brain, and in refinement of synapses

[Developmental potential of human fragmented embryos]

Introduction: The aim of this investigation was to study the developmental potential of human fragmented embryos to blastocyst stage and to evaluate the developed blastocysts in the term of size, cellularity, the type of inner cell mass [ICM], trophoetoderm [TE] and the number of dead cells

Material and Methods: Four to eight cell fragmented human embryos were obtained from the embryology lab of Royan Institute. Embryos were scored according to the degree of fragmentation, using invert microscope, into four groups [group I: embryos with least fragmentation, group II: embryos with small fragment among blastomers, group III: embryos with large fragment approximately in the size of blastomers and group IV: embryos with necrotic fragments]. Each group was cultured in rS2 medium and their development was recorded to the day six [embryos were cultured in rS1 for first 3 days].In this study the size and the quality of developed blastocyst and number of blastomer in the ICM and TE were evaluated. The number of dead cells in each blastocyst [apoptotic or necrotic] were counted using TUNEL staining and the results were analyzed statistically by chi2 and ANOVA

Results: High percentage of embryos from group IV had developmental block at 2- 4 cell stage. In addition, a high percentage of embryos from all groups stopped their development at 8-cell to morolla stage. The rate of blastocyst formation of embryos from group I and II was higher than group III and IV. The results showed that size of embryos from group IV was statistically lower than groups I and II. Blastocyst quality [the number of blastomers in ICM] of embryos from group I and II was better than group III and IV. Differential staining results suggested that the ratio of ICM to TE in embryos from each group is not statistically different compared to other groups. But the mean total number of blastomer and the mean number of ICM and TE of embryos from groups I and II was higher than the other two groups. The results of TUNEL staining showed that embryos with high number and size of fragmentation at 4-cell stage had a high number of apoptotic and necrotic cell

Conclusion: Embryos with high fragmentation have reduced developmental potential, blastocyst size, low quality blastocyst, low quality ICM and TE, and low number of total cell number. In addition the number of dead cell was higher