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Acquisition and Expansion of Adult Rat Bone Marrow Multipotent Mesenchymal Stromal Cells

Abstract

This study was initiated in order to test a mini-invasive method of mesenchymal stem/progenitor cells (MS/PCs) isolation from a rat bone marrow (BM), and subsequently their expansion, differentiation, and evaluation of their immunophenotypic characteristics; and later their preservation as donor cells in an optimal condition for potential autotransplantation. The study group comprised of 6 adult male Sprague-Dawley (S-D) rats, weighing 480—690 g. The rats were anaesthetised by isoflurane with room air in a Plexiglas box and maintained by inhalation of a mixture of isoflurane and O2. Their femurs were surgically exposed and their diaphyses double-trephined. Then BM cells were flushed out by saline with heparin and aspirated into a syringe with a solution of DMEM (Dulbecco’s modified eagle’s medium) and heparin. The mononuclear cells from the BM were isolated by centrifugation and expanded in a standard culture medium supplemented with ES-FBS (es-cell-qualified foetal bovine serum), L-glutamine and rh LIF (recombinant human leukemia inhibitory factor). Following 14 days of passaging cultures, the cells were split into 2 equal parts. The first culture continued with the original medium. The second culture received additional supplementation with a human FGFβ (fibroblast growth factor beta) and EGF (epidermal growth factor). The populations of these cells were analysed by light-microscopy, then the mean fluorescence intensities (MFIs) of CD90 and Nestin were evaluated by a tricolour flow cytometry using monoclonal antibodies. The type of general anaesthesia used proved to be appropriate for the surgical phase of the experiments. All rats survived the harvesting of the BM without complications. The total number of mononuclear cells was 1.5—4.0 × 106 per sample and the proportion of CD90/Nestin expressing cells was < 1 %. Following 14 days of expansion, the cells became larger, adherent, with fibrillary morphology; the proportion of cells expressing CD90/Nestin increased to almost 25 %, i. e. they earned basic phenotypic characteristics of MSCs. Throughout the further cultivation a gradual decrease of the CD90/Nestin expression occurred. This suggested that the suitability of rat bone marrow derived MS/PCs for replacement therapy would probably be the highest between days 12—15 of cultivation and then would diminish.

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Oxidative stress induced by the Fe2+/ascorbic acid system or model ischemia in vitro: effect of carvedilol and pyridoindole antioxidant SMe1EC2 in young and adult rat brain tissue

Oxidative stress induced by the Fe2+/ascorbic acid system or model ischemia in vitro: effect of carvedilol and pyridoindole antioxidant SMe1EC2 in young and adult rat brain tissue

New effective strategies and new highly effective neuroprotective agents are being searched for the therapy of human stroke and cerebral ischemia. The compound SMe1EC2 is a new derivative of stobadine, with enhanced antioxidant properties compared to the maternal drug. Carvedilol, a non-selective beta-blocker, possesses besides its cardioprotective and vasculoprotective properties also an antioxidant effect. We compared the effect of carvedilol and SMe1EC2, antioxidants with a similar chemical structure, in two experimental models of oxidative stress in young and adult rat brain tissue. SMe1EC2 was found to improve the resistance of hippocampal neurons to ischemia in vitro in young and even in 18-month-old rats and inhibited formation of protein carbonyl groups induced by the Fe2+/ascorbic acid pro-oxidative system in brain cortex homogenates of young rats. Carvedilol exerted a protective effect only in the hippocampus of 2-month-old rats and that at the concentration 10-times higher than did SMe1EC2. The inhibitory effect of carvedilol on protein carbonyl formation induced by the pro-oxidative system was not proved in the cortex of either young or adult rats. An increased baseline level of the content of protein carbonyl groups in the adult versus young rat brain cortex confirmed age-related changes in neuronal tissue and may be due to increased production of reactive oxygen species and low antioxidant defense mechanisms in the adult rat brain. The results revealed the new pyridoindole SMe1EC2 to be more effective than carvedilol in neuroprotection of rat brain tissue in both experimental models involving oxidative stress.

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Alterations in intestinal and liver histomorphology and basal hematological and biochemical parameters in relation to different sources of dietary copper in adult rats

Abstract

Copper (Cu) is required for all basic biochemical and physiological processes. The aim of this study was to evaluate the effects of different sources of dietary Cu on the histomorphometry of liver and jejunal epithelium in adult rats. Male 12-week-old rats were used in a 12-week experiment. The control diet provided the required Cu level from sulfate, and other two diets were supplemented with Cu as a glycine complex at 75% and 100% of daily requirement. Basal hematological and plasma biochemical analyses were also performed. There was no effect of Cu supplementation on the liver weight and the plasma and liver Cu concentration. Histomorphometric analysis of liver tissue showed an increase in the collagen amount and intracellular space in the group supplemented with Cu amino acid. Cu given in the organic form at 100% of daily requirement decreased the muscular and submucosa layer and the crypt depth. In turn, organic copper given at 75% of daily requirement did not influence the intestinal morphology. Dietary Cu given to adult rats as copper sulfate or a glycine complex meeting 100% of the daily requirement appears to be less harmful with regard to intestinal epithelium than when given as a glycine complex at 100% of daily requirement.

Open access
Effect Of High-Fructose Solution On Body Weight, Body Fat, Blood Glucose And Triglyceride Levels In Rats

Summary

In Europe, as well as in Bulgaria, consumption of soft drinks and confectionery has increased during the last three decades and is partly responsible for the epidemic-like increase in obesity. These foods, originally sweetened by sucrose, are now sweetened by other caloric sweeteners such as fructose. In this study we investigated the effect of an eight-week intake of 20% fructose solution on body weight in rats. Two adult rat groups (aged 120±6 days) of Wistar line were studied: a Control group (C; n=10; 5 male and 5 female rats) received water and standard rodent chow, and a Fructose group (F; n=12; 6 male and 6 female rats) who received 20% fructose-in-drinking-water solution and regular rodent chow. All animals were weighed and measured (nose to anus length), and the Lee index (equivalent of BMI in rats) was calculated. Body fat was also analyzed. As indicators of increased caloric intake of the Fructose group we investigated glucose, triglycerides and cholesterol (total, HDL and LDL) in blood. In conclusion, consumption of fructose solution in rats resulted in increased body weight, length and measured body fat, increased blood glucose, total cholesterol and triglycerides in the Fructose group, as compared to the controls.

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Analgesic Effect of Local Infiltration of the Surgical Wound Containing Levobupivacaine, Ibuprofen, and Epinephrine in Rats Undergoing Laparotomy

Abstract

Background: Inappropriate use of drugs and their combinations for analgesics has made it difficult to determine the optimal drug combinations for pain mangement.

Aim: To reduce postoperative pain effectively and safely.

Materials and methods: Laparotomy was performed in an adult rat under isoflurane anesthesia. During surgery, the surgical wounds were infiltrated with 50 μL solution containing 0.3% w/v levobupivacaine, 2 mg/mL ibuprofen, and 8 mg/mL epinephrine (treatment group) over the sutured muscle wound before skin closing, and compared to infiltration of that of the normal saline (vehicle group). The 10-fold higher dose of the same combination of medications was injected systemically as a control. Postoperative pain assessed by rodent grimace scales scoring. One-way ANOVA following Dunnett multiple comparisons test was used at 95% of confidence level.

Results: There was decreased pain for the treatment group (p = 0.025, q = 4.527) and the control group (p = 0.031, q = 4.178) only 24 h after the end of the successful infiltration. The rodent GS scale scoring showed the fall in pain was started within three hours post-surgery in the treatment group. There was decreased pain in the treatment group (p = 0.048, q = 3.527) and the control group (p = 0.043, q = 3.891) only as compared to vehicle group 24 h after the end of the successful infiltration.

Conclusion: The infiltration of the surgical wound with levobupivacaine, ibuprofen, and epinephrine combination was effective in the healing of wounds after laparotomy.

Open access
A contribution to the knowledge of diet composition of the Barn Owl Tyto alba in the area of Pisa (Italy)

Abstract

We examined the pellets of the Barn Owl Tyto alba, collected in Pisa, Italy, in 2012. Altogether, 219 specimens of small mammals were found in 85 pellets. The Barn Owl diet was composed of ten species of small mammals, representing three different families (Muridae, Cricetidae, Soricidae). The main prey species was the Wood Mouse Apodemus sylvaticus, followed by the House Mouse Mus musculus and the Savi’s Pine Vole Microtus savii. While the smallest of the small mammals from the area, the Etruscan Shrew Suncus etruscus, was well represented in the pellets, some larger species of small mammals were not represented at all. The reason for such result may lie in the upper limit for our Barn Owl’s prey size. Results suggest that optimal prey weight for our Barn Owl may be between 26–75 g of body mass, however, the prey can be occasionally as heavy as almost 100 g, represented by adult Rat Rattus spp. Nevertheless, our results may not reflect the true hunting strategy of the Barn Owl, but the availability of a certain food item at one point in time.

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Vitamin E reduces sensory neuronal loss and improves nerve regeneration after sciatic nerve injury

References 1. Arvidsson J, Ygge J, Grant G. Cell loss in lumbar dorsal root ganglia and transganglionic degeneration after sciatic nerve resection in the rat. Brain Res. 1986; 373:15-21. 2. McKay Hart A, Brannstrom T, Wiberg M, Terenghi G. Primary sensory neurons and satellite cells after peripheral axotomy in the adult rat: timecourse of cell death and elimination. Exp Brain Res. 2002; 142:308-18. 3. Senoglu M, Nacitarhan V, Kurutas EB, Senoglu N, Altun I, Atli Y, Ozbag D. Intraperitoneal Alpha-Lipoic Acid to

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Lycopene supplementation prevents reactive oxygen species mediated apoptosis in Sertoli cells of adult albino rats exposed to polychlorinated biphenyls

References Anway MD, Folmer J, Wright WW, Zirkin BR. (2003). Isolation of Sertoli cells from adult rat testes: an approach to ex vivo studies of Sertoli cell function. Biol Reprod . 68 : 996-1002. Atessahin A, Karahan I, Turk G, Gur S, Yılmaz S, Ceribas AO. (2006a). Protective role of lycopene on cisplatin induced changes in sperm characteristics, testicular damage and oxidative stress in rats. Reprod Toxicol 21 : 42-47. Atessahin A, Turk G, Karahan I, Yilmaz S, Ceribasi AO, Bulmus O. (2006b

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11β Hydroxysteroid Dehydrogenase Type 2 in the Adrenal Gland by Testosterone Withdrawal of Adult Rats

protein expression in the adult rat testis following androgen withdrawal by ethane dimethanesulfonate. Folia Histochemica et Cytobiologica 2006;44(2):81-86. Plećas B, Pesić VP, Mirković D, Majkić-Singh N, Hristić M, Solarović T. Opposite effects of dexamethasone and ACTH on the adrenal cortex response to ethane dimethanesulphonate (EDS). Exp Toxicol Pathol 2001;53(1):31-4. Petrova E, Koeva Y, Bakalska M, Atanassova N, Davidoff M. Morphofunctional characteristics of rat adrenocorticocytes after treatment with ethane

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