Cell Journal (Yakhteh)
Year:1999 | Volume:1 | Issue:1|Papers Count:9

Introduction: Today, investigators pay attention to natural fluids (such as follicular and amniotic fluid) to use them as a medium or a supplement. Using human amniotic fluid (HAF) in some researches, not only improved in vitro development of preimplantation embryos but also significantly increased percentage of pregnancy. On the contrary, Gianaroli et al. have claimed there was no significant difference in fertilization, cleavage and pregnancy rates between HAF and control. Dorfmann et al. have also claimed HAF reduced pregnancy rate. Some investigators have worked on chick embryo amniotic fluid (CEAF). Blakwood et al and Ocampo et al. tansfered preimplantion mammalian embryo in amniotic cavity of developing chick embryo (CEAm) and indicated better mammalian embryo development. However, using aspirated CEAF by Blakewood et.al. didn't show significant difference compare to control medium. Since CEAF is more available, its aspration is not dangerous and does not cost much in comparison to HAF and syntetic culture media, and there is also controversy on this pointe, decided: 1) To evaluate the possibility effects of 20%, 50%, 100% inactivated and 100% active CEAF on the development of mouse embryo. 2) To investigate the differences between 6- and 10- days old CEAF on the development of mouse embryo. 3) To introduce CEAF as natural medium at least for the mouse embryo culture. Materials and metods: 6- and 10 - days old chick embryo amniotic fluid (6 - AF, 10- AF) were aspirated and two experiments were designed. Experiment one: 6 - AF was prepared as followed: Ham’s F - 10 + 20% inactivated AF (iAF20), Ham's F - 10 +50% inactivated AF (iAF 50), inactivated AF (iAF 100), active AF (AF 100), Ham’s F-10 + 10% albuminar -5 (SA:as control). Two-cell mouse embryo (Rondom-Bred swiss white Mice strain) was assigned to these five groups randomly. Experiment two: All of this procedure was repeated for 10- AF, as well. Results: Experiment one: In AF 100, iAF100 and iAF50 the percentage of total blastocyst (TB) was significantly higher than SA (87.5, 81.7, 82.1 vs 56.1 resp.)(P<0.001) Hatched blastocyst (HB) percentage was significantly higher in AF100 and iAF20compare to control (58,60.3 vs 22.5 resp.)   iAF20 iAF50 iAF100 iAF100 SA TB 72.2 82.1* 81.7* 87.5* 56.1* HB 60.3* 50.8 52.2 58 22.5 *:P<0.001  Experiment two: percentage of TB was significantly higher in AFlOO and iAFlOO compare to SA(82.3,73.6 vs 51.9 resp.) (P<0.001). HB percentage in AF100, iAF100 and iAF50 was significantly higher than SA (68,69.7,59.2 vs 30.3 resp.)(P<0.001).   iAF20 iAF50 iAF100 iAF100 SA TB 96.2 65.9 73.6* 82.3* 51.9* HB 51.8* 59.2 69.7* 68* 30.3 *:P<0.001  No significant difference was found between two experimental groups, 6-AF and 10-AF,and between different densities of CEAF ,as well. Conclusion: CEAF as a supplement or a natural medium could support the development of preimplantation mouse embryo.  

Introduction: in this study we examined the effect of ampullary and isthmic epithelial cell of golden hamster oviduct on N-Mary mouse embryo development during 4-days in vitro co - culture, and the effect of injected gonadotropins on the quality of co-culture. Materils and methods: Golden hamster oviducts were isolated 18-24h after treating the hamster with HCG. They were flushed with DMEM / Ham’s F - 12+FCS %10. According to presence of cumulus mass, the oviducts were classified into two categories: (i) responding to gonadotropins which contained cumulus mass, and (ii) non - responding to gonadotropins, which showed cumulus mass. Using collagenase ampullary (A) and isthmic (I) epithelil cells from each category were isolated and cultuered in medium. A monolayer culture system of hamster oviducts were estabilished. The late, 2- cell N-MARY mouse embryos were cultured for 4- days with T6+FCS %10 on the monoalyers (A-I) and T6+GCS. 10% as control group (C) in each category. Results: (i) After 96h hatching blastocyst in A, I and C were 64% , 50% and 36% respectively, which, significantly higher in co-culture groups (P<0.001 for A , P<0.05 for I).In addition during first 24h, embryo development in ampullary co - culture was better than isthmic co-cultures specially in ampulla (P<0.001 for A and P<0.05 for comarsion of A and I) (ii) After 96h ,more embroys developed to blastocyst in C group than A and I groups, (C= %58, A= %20, I=%27, P<0.001) Conclusion: Hamster oviduct epithelial cells which are specialized cells from ampullary region could improve the development of embryos from other species and this is greatly affected by response of hamster to gonadotropins.

Introduction: There are some internal and external factors in the development and supporty of joint. The role of movement on foint development could be study in three methods. A. In vitro limb buds culture B. Limb bud Graft to coelomic membrance. C. Neuromuscular function block. The development of the fourth metatarsophalyngeal joint of swiss mice was studied in normal circumstances and the absence of movement. Materials & Methods: In order to study the development of the fourth metatarsophalyngeal joint in absence of movement, mouse limb buds were separated from 15-days old embryso and cultured in Ham’s F -10 medium conditioned with 37˚C and 5% co2 for 1 to 6 days. The medium was renewed every 24h during incubation. After 6 days, specimens were fixed by Buin’s fluid, embedded in paraffin was, serially sectioned and stained with H & E. Results: The results indicated that, the mesenchymal cells of joint interzone were differentiated to fibroblast like cells and chondrocytes rather than losing to form joint space. Conclusion: It seems that the absence of movement in vitro conditions could effect the joint development and its intra – articular structures.

Introduction: A number of maternally inherited mitochondrial diseases with distinct clinical phenotypes have been associated with point mutations in mtDNA, all of which result in neurologic or neuromuscular disorders. Several studies showed that mutations in the tRNA genes of mtDNA could cause mitochondrial disease due to the decreased synthesis of mitochondrial DNA coded proteins. Materials and methods: A patient having clinical, biochemical, and histochemical abnormalities compatible with mitochondrial disease but without rearrangements in her mtDNA, was subjected to study for point mutations in the mitochondrial tRNA genes. Using molecular biology techniques such as PCR, RFLP and sequencing analysis. Results: A new mutation in mtDNA involving tRNASer(UCN) was identified in this patinet with muscle weekness and cardiomyopathy. This heteroplasmic T to C transition at position 7480 affects the anticodon loop at a highly conserved site and was not detected in control individuals. Analysis of mtDNA from blood cells and muscle showed that the mutation was present in both tissues. Levels of the mutant mtDNA in muscle and white blood cells were quantified and showed 73% and 30% heteroplasmy in the investigated tissues, respectively. The mutation was not present in the blood cells taken from the patient's relatives. Study of cross sectioned of the muscle fibres revealed the difference in the mutant content of the COX deficient and normal fibres in this patient. COX deficient fibres harboured more than 90% mutant mtDNA while the level of mutation in the COX normal fibres ranged from 9% to 68%. Conclusion: These data suggest that this mutation could well be the cause of clinical presentation of mitochondrial disease in this patient.

Introduction: With regarding the positive effects of low power Gallium Aluminium Arsenid laser (Laser) on some muscloskeletal disorders in this investigation effects of laser on open skin full thickness wound in rat by histological and biomechanical methods were evaluated. Materials & Methods: 46 male Sprague dawley rats randomly divided into control and experimental groups. Under general anaesthesia and sterile conditions on the skin of dorsal region of each rat one full thichness round wound were made. The day of wounding was considered day zero. From day 1 until day 4, day 7 and day 15 rats of experimental groupe received everyday laser radiation with a density of 1.2 J/cm2. At above mentioned day rats were killed by ether and 2 samples were fixed and prepared for routine histological study and stained with Hematoxylin and Eosin. The number of fibroblasts, macroghages, neutrophil, endothelial cells and blood vessels were counted. Biomechanical study was done and tensile strength of samples was calculated. Data were analysed by Mann Whitney U test method and p<0.05 were significant. Results: At experimental group the mean of fibroblast at day 7 and 15,the means of tensile strength at day4 and day 15 and the means of blood vessels at day 7 and of endothelium at day 15 were significantly higher. The mean of macrophages was significantly lower. Conclusion: Low power Gallium Aluminium Arsenide laser radiation on open full thickness skin wound of rats was significantly accelerated wound healing process.

Introduction: Concerning that third degree burn wounds have no real practical treatment other than invasive methods, this study reports on the therapeutic effects of a newly formulated ointment "fundermol" on accelerating the burn wound healing. Materials & Methods: Forty five male and female Albine-N-Mari rats weighing 160 to 180 g were anaesthesized with intraperitoneal injection of pentobarbital and burned by Steam in a way making third degree burn wound on their back 33 mm length in diameter. Then animals were randomly asigned to fundermol, silver sulfadiazine, and control groups. On post-wounding days 7, 14, and 28, the animals were sacrificed with ether and similar samples were excised in all groups. Epithelialization of wounds was evlauated in terms of hisotological analysis and morphometric assessment. Results: The statistical results indicate that Fundermol ointment increases the epithelialization of third degree burn wounds and leads to wound closure. We assume that the increase in vascularity of the wound bed and the support of the hair follicles as the result of the fundermol usage (24,25) can lead to a better epithelialization procedure. Also anti inflamatory and anti bacterial effects of the ointment may ease this procedure. Of course the exact mechanisms involving the therapeutic effects of this herbal ointment is yet to be studied. Conclusion: As fundermol ointment is a natural product and there is no report on the side effects of the drug components, these finidings may suggest a therapeutic clue in clinical management of third degree burn wounds.

Introduction: Fluorscence in situ hybridization (FISH) enables specific detection of unique sequences of varying length, chromosomal regions or entire chromosomes within metaphase or interphase cells. Recent developments in this technology permit the rapid mapping and ordering of DNA fragments on single metaphase chromosome bands. The technique of FISH incorporates several stages including: probe preparation and labelling, hybridization of probe with chromosome preparation and detection of signal, and imaging. Using these technique chromosomal alterations in ataxia telangiectasia lymphoblastoid cells which are highly sensitive to clastogenic and mutagnic effects of chemnical and physical agents were investigated. Materials and Methods: Normal and A-T cells were grown in RPMI – 1640 medium and irradiated at G2 and G1 phases of the cell cycle. After democolcine treatment and metaphase chromosome preparation, slides were prepared and FISH is performed for all samples using whole chromosome probe for chromosomes 5, 1, 4, 7 and 14. Fifty mitoses were analysed for each sample by Zeiss fluorescent microscope attached to a computerized programme. Results: Analysis of normal and A – T mitoses before and after irradiation revealed a very low frequency of chromosomal abnormalities, for specific chromosomes 5 painted in  this study. However, more aberration was found in A-T cells of varios chromosomal rearrangements such as translocation, trisomy, Robertsonian translocation and chromatid type breaks. Conclusion: In routin cytogentic methodologies visualisation of various chromosomal rearrangements is not always possible in a single preparation. Although detecton sensitivity of FISH is limited to the specific chromosomes, but as seen for A – T cells, FISH can be considered as one of the most effective method for visualization of various chromosomal alterations. This technique can have wide application not only in human genome mapping and the genome of other organisms, but also in clinical cytogenetics, somatic cell genetics, cancer diagnosis and gene expression studies.

Introduction: Fusimotor neurons innervate intrafusal muscle fibers within muscle spindles. They are of at least three different types: static and dynamic axons innervating static and dynamic bag fiber respectively. Different areas in central nervous system are known to influence their activity. In present study, cortical control of the gamma motoneurons of tenuissimus muscle spindle in eight anaesthetized cats was investigated using direct visual method. Materials and Methods: Cats of either sex, weighing 2-3.8 kg were anaesthetized with sodium pentobarbitone (45 mg/kg), tracheotomized and intubated, obturator and femoral nerves cut and all the hindlimb muscles except tenuissimus denervated (right sciatic nerve also eat). Muscle spindles uncovered microsurgically and placed in the bath overviewed by a phillips camcorder. Right sensorimolor cortex was then exposed and a paraffin pool was made using skull skin. 0.3 to 3 mA current were applied, both anodal and cathodal, within a five second period of a 12 second stimulation window. Spontaneous and evoked intrafusal movements were monitored and taped for furthur analysis. Results: The findings confirmed the evidence for independent cortical control of different types of static gamma motoneurons and revealed a topographical mapping of the sensorimotor cortex in relation to the type of gamma motoneurons recruited, either static or dynamic. Static effects were elicited following stimulation of a wide area across the sensorimotor cortex, the postcruciate dimple being almost at the center. Within that region, a "dynamic area" was identified from which dynamic effects were clearly elicited during stimulation. Conclusion: Different types of static gamma axons and dynamic gamma axon can be controlled independently on cerebral activation. The state of anaesthesia was crucial in obtaining reproducible results and its variation could alter the fusimotor effect from static to dynamic or even from excitation to inhibition.