scholarly journals In vitro mature embryo culture protocol of einkorn (Triticum monococcum ssp. monococum) and bread (Triticumaestivum) wheat under boron stress

2021 ◽  
Author(s):  
Ferdi Ağıl ◽  
Mehmet Örgeç ◽  
Fatma Pehlivan Karakaş ◽  
Sandeep Verma Kumar ◽  
Nusret Zencirci
Keyword(s):  
Bread Wheat ◽  
Culture Media ◽  
Mature Embryo ◽  
Wheat Breeding ◽  
Triticum Monococcum ◽  
Control Group ◽  
Einkorn Wheat ◽  
Mature Embryos ◽  
Different Doses

Abstract Mature embryos of einkorn (Triticum monococcum ssp. monococcum) and bread (Triticum aestivum) wheat were used for callus induction on media containing four (0, 1, 2 and 4 mg L− 1) different doses of 2,4-D and dicamba supplemented with under five (0, 6.2, 12.4, 24.8, and 37.2 mg L− 1) different boron stresses. The obtained callus was transferred to culture media with three (0, 0.5, and 2 mg L− 1) different BAP doses with five boron stresses for regeneration. The heaviest callus weight in einkorn wheat was in culture media with 1 mg L− 1 dicamba and 6.2 mg L− 1 (3.71 ± 0.13 g). Bread wheat had the heaviest callus weight on culture media with 4 mg L− 1 dicamba and 12.4 mg L− 1 (3.46 ± 0.40 g). Callus diameters were observed as the highest in culture media with 1 mg L− 1 and 12.4 mg L− 1 (1.10 ± 0.31) for einkorn and 4 mg L− 1 dicamba with 6.2 mg L− 1 boron (1.22 ± 0.27 cm) for bread wheat. Regeneration capacity was highest in control group with 6.2 mg L− 1 for both wheat genotypes (einkorn, 71.33% ± 11.78 and bread, 65.33% ± 10.80). The highest plantlet numbers were in only 2 mg L− 1 BAP (2.92 ± 0.88) for einkorn wheat and 0.5 mg L− 1 BAP supplemented with 6.2 mg L− 1 boron (3.71 ± 1.12) for bread wheat. This indirect regeneration protocol using mature embryos of einkorn and bread wheat under boron stresses may be useful for wheat breeding studies.

Evaluation of Annona muricata (Graviola) leaves activity against experimental trichinellosis: in vitro and in vivo studies

2021 ◽  
Vol 95 ◽  
Author(s):  
E.S. El-Wakil ◽  
H.F. Abdelmaksoud ◽  
T.S. AbouShousha ◽  
M.M.I. Ghallab
Keyword(s):  
Culture Media ◽  
Trichinella Spiralis ◽  
Drug Effects ◽  
In Vivo Studies ◽  
Control Group ◽  
Annona Muricata ◽  
Group I ◽  
Small Intestinal

Abstract Our work aimed to evaluate the possible effect of Annona muricata (Graviola) leaf extract on Trichinella spiralis in in vitro and in vivo studies. Trichinella spiralis worms were isolated from infected mice and transferred to three culture media – group I (with no drugs), group II (contained Graviola) and group III (contained albendazole) – then they were examined using the electron microscope. In the in vivo study, mice were divided into five groups: GI (infected untreated), GII (prophylactically treated with Graviola for seven days before infection), GIII (infected and treated with Graviola), GIV (infected and treated with albendazole) and GV (infected and treated with a combination of Graviola plus albendazole in half doses). Drug effects were assessed by adults and larvae load beside the histopathological small intestinal and muscular changes. A significant reduction of adult and larval counts occurred in treated groups in comparison to the control group. Histopathologically, marked improvement in the small intestinal and muscular changes was observed in treated groups. Also, massive destruction of the cultured adults’ cuticle was detected in both drugs. This study revealed that Graviola leaves have potential activity against trichinellosis, especially in combination with albendazole, and could serve as an adjuvant to anti-trichinellosis drug therapy.

scholarly journals Cilengitide Inhibits Neovascularization in a Rabbit Abdominal Aortic Plaque Model by Impairing the VEGF Signaling

2021 ◽  
Vol 2021 ◽  
pp. 1-18
Author(s):  
Fawang Zhu ◽  
Shuai Yuan ◽  
Jing Li ◽  
Yun Mou ◽  
Zhiqiang Hu ◽  
...  
Keyword(s):  
Umbilical Vein ◽  
Tube Formation ◽  
Control Group ◽  
In Vitro Experiments ◽  
Aortic Plaque ◽  
Abdominal Aortic ◽  
Aortic Plaques ◽  
Different Doses

Background. Cilengitide is a selective αvβ3 and αvβ5 integrin inhibitor. We sought to investigate the effect of cilengitide on the neovascularization of abdominal aortic plaques in rabbits and explore its underlying antiangiogenic mechanism on human umbilical vein endothelial cells (HUVECs). Materials and Methods. For the in vivo experiment, the abdominal aortic plaque model of rabbits was established and injected with different doses of cilengitide or saline for 14 consecutive days. Conventional ultrasound (CUS) and contrast-enhanced ultrasound (CEUS) were applied to measure the vascular structure and blood flow parameters. CD31 immunofluorescence staining was performed for examining neovascularization. Relative expressions of vascular endothelial growth factor (VEGF) and integrin of the plaque were determined. For in vitro experiments, HUVECs were tested for proliferation, migration, apoptosis, and tube formation in the presence of different doses of cilengitide. Relative expressions of VEGF, integrin, and Ras/ERK/AKT signaling pathways were determined for the exploration of underlying mechanism. Results. CEUS showed modestly increased size and eccentricity index (EI) of plaques in the control group. Different degrees of reduced size and EI of plaques were observed in two cilengitide treatment groups. The expressions of VEGF and integrin in the plaque were inhibited after 14 days of cilengitide treatment. The neovascularization and apoptosis of the abdominal aorta were also significantly alleviated by cilengitide treatment. For in vitro experiments, cilengitide treatment was found to inhibit the proliferation, migration, and tube formation of HUVECs. However, cilengitide did not induce the apoptosis of HUVECs. A higher dose of cilengitide inhibited the mRNA expression of VEGF-A, β3, and β5, but not αV. Lastly, cilengitide treatment significantly inhibited the Ras/ERK/AKT pathway in the HUVECs. Conclusions. This study showed that cilengitide effectively inhibited the growth of plaque size by inhibiting the angiogenesis of the abdominal aortic plaques and blocking the VEGF-mediated angiogenic effect on HUVECs.

183 ADDITION OF DIPHENYLENEIODONIUM OR DEHYDROEPIANDROSTERONE TO CULTURE MEDIA INHIBITS REACTIVE OXYGEN SPECIES PRODUCTION DURING THE EARLY CLEAVAGE STAGES OF IN VITRO-PRODUCED PORCINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 208
Author(s):  
N. W. K. Karja ◽  
K. Kikuchi ◽  
M. Ozawa ◽  
M. Fahrudin ◽  
T. Somfai ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Nadph Oxidase ◽  
Culture Media ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reactive Oxygen ◽  
Blastocyst Stage ◽  
Control Group ◽  
Ros Production ◽  
Oxygen Species

Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), an enzyme required to catalyze the oxidation of NADPH to NADP during the metabolism of glucose via the pentose phosphate pathway (PPP), was considered as contributing to intracellular reactive oxygen species (ROS) production. Production of superoxide anion and H2O2 via NADPH oxidase has been reported on a rabbit blastocyst surface (Manes and Lai 1995 J. Reprod. Fertil. 104, 69–75). The objective of this study was to examine the effects on in vitro development and intracellular ROS content after the addition of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, or dehydroepiandrosterone (DHEA), an inhibitor of glucose-6-phosphate dehydrogenase (G6PDH), to culture medium during the early embryonic development of in vitro-produced (IVP) porcine embryos. To confirm that these inhibitors lead to reduction in NADPH concentration in the embryo and hence likely to be inhibiting the PPP, a brilliant cresyl blue (BCB) test was performed on Day 2 (the day of insemination = Day 0) of culture. Porcine cumulus–oocyte complexes were matured and fertilized in vitro as described previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Prezumptive zygotes were then cultured in NCSU-37 supplemented with 5.5 mM glucose and DPI at concentrations of 0.5 or 1 nM or DHEA at concentrations of 10 or 100 �M (DPI-0.5, DPI-1, DHEA-10 and DHEA-100 groups, respectively) from Day 0 to Day 2 of culture. All of the embryos were cultured subsequently until Day 6 in NCSU-37 supplemented with only 5.5 mM glucose. Data were analyzed by ANOVA. On Day 6, the development to the blastocyst stage of embryos in DPI-0.5, DPI-1, DHEA-10, and DHEA-100 groups were 16.1, 17.6, 16.1, and 19.5%, respectively, which were not significantly different from that of the control group (17.5%) (n d 165 per group, 5 replicates). However, the mean cell number in blastocysts derived from DPI-1, DHEA-10, and DHEA-100 groups (40.8 � 2.3, 39.3 � 1.7, and 42.5 � 2.7, respectively) was significantly higher (P < 0.01) than those in the control (33.4 � 1.6) and DPI-0.5 (32.7 � 1.6) groups. At 20 min after an exposure to BCB, the percentage of BCB+ embryos in DPI-1, DHEA-10, and DHEA-100 groups (73.8, 79.9, and 77.8%, respectively) were significantly higher (P < 0.01) than those in the control and DPI-0.5 groups (42% and 53.9%, respectively) (n = 81-92 per group, 6 replicates), indicating that these two inhibitors effectively induce the reduction of NADPH concentration in the embryos. Moreover, the addition of DPI at 1 nM or DHEA at 10 or 100 �M significantly decreased the H2O2 content of Day 2 embryos as compared with control embryos (n = 48-53 per group, 7 replicates). These results suggest that the addition of either DPI or DHEA to the medium during the first 2 days of culture did not impair the development of the embryos to the blastocyst stage. Decrease of cellular ROS production in Day 2 embryos in this study is interpreted as a result of inhibition of the NADPH oxidase by DPI or of the G6PDH by DHEA.

60 IMPROVEMENT IN EFFICIENCY AND QUALITY OF CLONED AND PARTHENOGENETIC PORCINE EMBRYOS BY AMINO ACIDS

2007 ◽  
Vol 19 (1) ◽  
pp. 147
Author(s):  
H. T. Lee ◽  
J. M. Jang ◽  
S. H. Lee ◽  
M. K. Gupta
Keyword(s):  
Amino Acids ◽  
Culture Media ◽  
Cell Number ◽  
Control Group ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate

In vitro production of cloned porcine embryos by somatic cell nuclear transfer (SCNT) has become routine in several laboratories but the efficiency and quality of the resultant blastocysts remains sub-optimal. Cloned porcine blastocysts show low cell number, high fragmentation rate, and apoptosis which results in lower pregnancy rates upon embryo transfer. Earlier we reported that supplementation of culture media with amino acids benefit pre-implantation embryo development of in vivo- as well as in vitro-fertilized porcine embryos (Koo et al. 1997 Theriogenology 48, 791–802). This study evaluated how exogenous amino acids could affect pre-implantation development and quality of cloned or parthenogenetic porcine embryos. The effects of commercially available amino acids, referred to as Eagle's non-essential amino acids (NEAA), added or not added (control) to NCSU23 medium containing fatty acid-free BSA were studied. Oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro and parthenogenetically activated (PA) or nuclear-transferred with fetal fibroblasts (SCNT), as described earlier (Uhm et al. 2000 Mol. Reprod. Dev. 57, 331–337). At 168 h post-activation, blastocysts were harvested for assessment of embryo quality by TUNEL labeling, Hoechst 33342 staining, and gene expression analysis. Results showed that, in the PA group, the cleavage rate was not affected by the supplementation of NEAA. However, the blastocyst rate was significantly improved when NEAA was present in the medium compared to that of the control group (38.9 ± 0.3 vs. 27.5 ± 0.3%, respectively) throughout the culture period. The supplementation during the pre-compaction period alone gave better results than during the post-compaction period alone (59.5 ± 0.9 vs. 33.4 ± 0.3%, respectively). In the SCNT group, however, both cleavage (73.6 ± 0.2 vs. 64.2 ± 0.4%) and blastocyst rate (18.7 ± 0.2 vs. 13.8 ± 0.3%) were improved by NEAA supplementation. Furthermore, these blastocysts had higher hatching ability (30.0 ± 1.8 vs. 14.6 ± 4.9%) than those of control group (P < 0.05). Supplementation of NEAA also increased the mean nuclei number of PA-derived (76.1 ± 4.9 vs. 66.5 ± 3.3) as well as SCNT-derived (43.1 ± 2.6 vs. 31.8 ± 1.9) blastocysts and reduced the time during which blastocysts formed. TUNEL assay revealed that incidence of nuclear fragmentation and apotosis was reduced by NEAA. Real-time qRT-PCR for Bax and Bcl-XL transcripts revealed that the relative abundance of Bax was reduced while that of Bcl-XL was increased. These effects were more pronounced when NEAA was present during the pre-compaction period alone. Thus, our data suggest that NEAA improves the yield and quality of cloned porcine embryos by enhancing blastocyst expansion and positively modulating the total cell number and apoptosis. These data may have implications for understanding the nutritional needs of cloned porcine embryos produced in vitro and for optimizing the composition of culture media to support their development. This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.

113 EFFECT OF PLANT PROTEIN ON DEVELOPMENT AND QUALITY OF CULTURED IN VITRO PORCINE ZYGOTES

2009 ◽  
Vol 21 (1) ◽  
pp. 157 ◽  
Author(s):  
B. Gajda ◽  
I. Grad ◽  
Z. Smorag
Keyword(s):  
Serum Albumin ◽  
Culture Media ◽  
Quality Criteria ◽  
Cell Number ◽  
Developmental Competence ◽  
Plant Protein ◽  
Control Group ◽  
Group 1

Basic culture media are usually supplemented with serum albumin or serum, which contain amino acids that play an important role as energy sources, osmoregulators, and pH stabilizers. However, the presence of undefined serum in culture media introduces a variation from batch to batch and increases viral or prion contamination risk. The aim of the present study was to investigate the possibility of using plant protein substitute (PP) in place of bovine serum albumin (BSA) during in vitro culture of porcine zygotes. The PP is a mixture of several plant proteins and soya lecithin prepared using a high pressure homogenization process. The experiment was done on pig zygotes obtained surgically from superovulated gilts at 24–26 h after insemination. Morphologically normal zygotes were cultured in vitro in 5% CO2 in air at 39° in NCSU-23 medium supplemented with: 0.002 g mL–1 (group 1), 0.004 g mL–1 (group 2), 0.008 g mL–1 (group 3) PP or 0.004 g mL–1 BSA (control group). Embryo quality criteria were developmental competence (cleavage, morula and blastocyst rates), total cell number per blastocyst and degree of apoptosis as assessed by TUNEL method. Results were analyzed by Chi-square test. There were no differences in cleavage rates on Day 2 between zygotes cultured in NCSU-23 medium supplemented with PP (86.0, 88.0, 84.8; group 1 to 3, respectively) and BSA (91.0%, control group). Culture with 0.008 g mL–1 PP increased morula (85.7%) and blastocyst (69.2%) production as compared with control (75.0% and 56.3%, respectively; P < 0.05) and 0.002 g mL–1 PP (79.5% and 51.8%, respectively; P < 0.05). The mean number of cells in Day 7 blastocysts cultured in NCSU-23 medium + 0.004 g mL–1 BSA was lower (P < 0.05) than in NCSU-23 + 0.004 g mL–1 PP (39.1 v. 43.7, respectively). The blastocysts cultured in NCSU-23 medium + 0.002 g mL–1 PP had higher average number of apoptotic nuclei (13.0) as compared with the control (6.5) and 0.004 g mL–1 PP (6.9). In conclusion, this study suggest the positive effect of PP on development in vitro of porcine zygotes to the morula/blastocyst stage. However, further studies are required to determine the quality of the embryos cultured with PP. This study was supported by Scientific Net of Animal Reproduction Biotechnology.

334 EFFECT OF A GROWTH MEDIUM DURING IVM ON EMBRYO DEVELOPMENT OF PREPUBERTAL EWE OOCYTES

2010 ◽  
Vol 22 (1) ◽  
pp. 323 ◽  
Author(s):  
M. G. Catalá ◽  
D. Izquierdo ◽  
R. Romaguera ◽  
M. Roura ◽  
M. T. Paramio
Keyword(s):  
Ascorbic Acid ◽  
Embryo Development ◽  
Growth Medium ◽  
Culture Media ◽  
Sperm Concentration ◽  
Control Group ◽  
Developmental Potential ◽  
Cresyl Blue ◽  
Maturation Medium

The aim of this study was to assess the effect of an in vitro growth medium (De Wu et al. 2006 Biol. Rep. 75, 547-554) in prepubertal ewe oocytes selected by the brilliant cresyl blue (BCB) test. Prepubertal ewe oocytes were recovered by slicing ovaries of slaughtered animals and immediately exposed during 1 h to 13 μM BCB and classified according to their cytoplasm coloration (Rodriguez-Gonzalez E et al. 2002 Theri- ogenology 57(5), 1397-1409): BCB+ (blue cytoplasm, hypothetically grown oocytes) and BCB- (uncolored cytoplasm, hypothetically growing oocytes). Uncolored oocytes (BCB-) were matured using three culture media: growth medium (GM: TCM-199, 0.04 μg mL-1 FSH, 0.04 μg mL-1 LH, 0.004 μg mL-1 estradiol, 100 μg mL-1 ascorbic acid, and 5 μL mL-1 ITS: insulin transferrin selenium), conventional maturation medium (CM: TCM-199, 10 μg mL-1 FSH, 10 μg mL-1 LH and 1 μg mL-1 estradiol) and modified maturation medium (MM: CM with the addition of 100 μg mL-1 ascorbic acid and 5 μL mL-1 ITS). Oocytes were matured in GM for 12 h and then separated into 2 groups, CM (GM+CM) and MM (GM+MM) for another 12 h of maturation. Two extra groups of BCB- oocytes were directly cultured for 24 h in CM or MM media (BCB-/CM and BCB-/MM). Colored oocytes (BCB+) and a control group (oocytes not exposed to BCB) were matured for 24 h in CM. All groups were cultured at 38.5°C and 5% CO2 in humidified atmosphere. Fertilization took place in SOF medium supplemented with 10% of estrous sheep serum during 20 h with a sperm concentration of 1 × 106 spermatozoa/mL. Presumptive zygotes were cultured for 8 days in SOF with 10% FCS at 38.5°C, 5% CO2 and 5% O2. Results are shown in Table 1. The percentage of morula plus blastocyst obtained from BCB - oocytes was significantly increased in oocytes exposed to growth medium (containing ITS, ascorbic acid and low hormone concentrations; groups GM+CM and GM+MM) for the first 12 h. An increasing tendency has also been observed in blastocyst yield in these two groups. Regarding maturation rate, no differences were found in all groups (data not shown). In conclusion, as De Wu et al. (2006) showed in prepubertal gilts, we also achieved some improvements in embryo development of growing oocytes when the first 12 h of maturation took place in a growth medium. However, embryo developmental potential of BCB- oocytes is still lower compared with that of BCB+ oocytes. Table 1.Effect of GM on embryo development of BCB- oocytes Grant sponsor Spanish Ministry of Science and Innovation.Code: AGL2007-60227-CO2-01

314 MELATONIN ON IN VIVO AND IN VITRO MATURATION OF MOUSE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 246 ◽  
Author(s):  
H. Fernandes ◽  
L. Schefer ◽  
F. C. Castro ◽  
C. L. V. Leal
Keyword(s):  
Ovarian Stimulation ◽  
In Vitro Maturation ◽  
F1 Hybrids ◽  
Saline Control ◽  
Control Group ◽  
Mouse Oocytes ◽  
Maturation Rate ◽  
Different Doses

Melatonin is a pineal hormone related to the control of the circadian cycle, besides the reproductive seasonality of some animal species, and has shown positive effects on oocyte maturation and embryo development. The aim of this study was to assess the effects of melatonin on in vivo and in vitro maturation of mouse oocytes. Female F1 hybrids (C57BL/6 × CBA; n = 8 per group/treatment) were used in 3 different treatments (trt) groups: (I) in vivo trt: mice received 2 different doses of melatonin injections, 10 and 20 mg kg–1 per IP including a saline control dose (0 mg kg–1 per IP) for 4 days along with ovarian stimulation trt of 5 IU of eCG IP, followed by 5 IU of hCG IP 48 h later, and cumulus-oocyte complexes (COC) were collected 16 h after hCG; (II) mice received a similar in vivo melatonin trt, but ovarian stimulation trt was only 5 IU of eCG, no hCG, and COC were collected after 48 h and subsequently matured in vitro with 0.5 µg mL–1 of FSH for 16 h; (III) in vitro maturation of oocytes: COC were collected 48 h after 5 IU of eCG and maturated in the presence of 3 different doses of melatonin (10–9, 10–6, and 10–3 M) or 0.5 µg mL–1 of FSH (control) for 16 h. In vitro-maturing oocytes were in incubated at 37°C, 5% CO2, and 95% humidity. Maturation rates were evaluated according to the presence of the first polar body under an inverted microscope. Statistical analyses were performed by ANOVA followed by Tukey's test (4 replicates). In the first treatment, 20 mg kg–1 of melatonin showed the highest in vivo maturation rate, 80.3% (61/76), while 10 mg kg–1 of melatonin was 62.4% (53/85) and the saline control group was 69.4% (77/111), but differences were not significant (P > 0.05). For in vitro maturation of oocytes from animals previously treated with melatonin, the 10 mg kg–1 of melatonin group had the highest maturation rate, 53.2% (99/186), in comparison with the saline and 20 mg kg–1 of melatonin groups, which showed 46.6 (88/189) and 39.0% (85/218), respectively; again, no differences were detected (P > 0.05). In the last treatment, the maturation rates increased from 48.9 (43/88) to 53.7 (51/95) and 56.0% (56/100) as the melatonin concentrations decreased from 10–3, 10–6, and 10–9 M, respectively. The control group had the highest rate of 57.3% (55/96), but no statistical differences were observed (P = 0.706). In conclusion, under the conditions studied, melatonin was unable to improve the maturation rate neither after in vivo nor in vitro treatment. However, during in vitro maturation, melatonin alone was as efficient as FSH in promoting maturation in murine oocytes, indicating its potential effect on stimulating meiosis. Therefore, the role of melatonin in stimulating meiosis needs further investigation.Acknowledgments to FAPESP for fellowship (HF) and funding (CLVL).

scholarly journals COMPARISON OF THE AMOUNT OF APICAL EXTRUSION OF BACTERIA FOLLOWING THE USE OF DIFFERENT INSTRUMENTATION TECHNIQUES – AN IN VITRO STUDY

2011 ◽  
Vol 01 (04) ◽  
pp. 27-32
Author(s):  
Mithra N. Hegde ◽  
Snehal Thatte
Keyword(s):  
Culture Media ◽  
In Vitro Study ◽  
Control Group ◽  
Group Iii ◽  
Root Canals ◽  
Apical Extrusion ◽  
Significant Difference ◽  
Group Ii ◽  
Group 1

Abstract Objectives: The objective of this study is to compare the amount of extrusion of bacteria beyond the apical foramen after instrumentation with Crown down and Step-back techniques using a manual and engine driven nickel-titanium instruments Materials and Methods: Seventy-five mandibular premolars with similar dimensions were used for the study. Access cavities prepared and root canals contaminated with a suspension of Enterococcus faecalis. The contaminated teeth were then divided into three experimental groups. Group 1(Crowndown group) divided into two: Group 1–A Hand files: root canals were instrumented using K-files and Group 1B – Rotary files: root canals were instrumented using ProTaper instruments. Group II (Step-back group) divided into two: Group II A– Hand files: root canals were instrumented using K-files and group II B–Rotary files: the root canals were instrumented using Light Speed LSX instruments. Group III (control group): no instrumentation was done.Bacteria were extruded after preparation were collected into vials, microbiological samples were incubated in culture media for 24hrs. The CFUs were determined for each sample. The data obtained was analyzed using the Kruskal-Wallis one way analysis of variance and Mann-Whitney U-tests. Result: There was a significant difference in the amount of bacteria extruded by both Crowndown and Step-back. The Step- back hand method extruded significantly more bacteria when compared with Crowndown hand technique. Conclusion: All instrumentation techniques extruded intracanal bacteria apically. There was a significant difference in both the engine driven instrumentation techniques, while the hand instrumentation by Step-back extruded more bacteria.

scholarly journals Role of TNF-α among inflammatory molecules secreted by injured astrocytes in the modulation of in vitro neuronal networks

2020 ◽  
Author(s):  
Joséphine Lantoine ◽  
Anthony Procès ◽  
Agnès Villers ◽  
Sophie Halliez ◽  
Luc Buée ◽  
...  
Keyword(s):  
Neuronal Networks ◽  
Culture Media ◽  
Polymerization Process ◽  
Mortality And Morbidity ◽  
Neuronal Viability ◽  
Tnf Α ◽  
Inflammatory Molecules ◽  
Different Doses

AbstractTraumatic brain injury (TBI) remains one of the leading causes of mortality and morbidity worldwide. Despite its high prevalence and extensive efforts to develop neuroprotective therapies, effective treatments for TBI are still limited. Among important neuronal damages, TBI induces structural and functional alterations of astrocytes, the most abundant cell type in the brain. Injured astrocytes respond in diverse mechanisms that result in reactive astrogliosis and are involved in the physiopathological mechanisms of TBI in an extensive and sophisticated manner. The establishment of effective neuroprotective treatments for TBI requires to better understand the complex biochemical interactions between activated astrocytes and neurons that contribute to the secondary injury. To address this challenge, we studied in vitro the role of mechanically injured astrocytes on the growth and synaptic connections of cortical neuronal networks of controlled architectures grown on well-defined protein micropatterns. Astrocytes were cultivated on elastic membranes and mechanically activated by stretching cycles. The culture media of healthy or activated astrocytes was then introduced on neuronal networks. We analyzed the neuronal viability, the neurite growth and the synaptic density of neuronal networks to understand the role of the inflammatory molecules secreted by mechanically activated astrocytes. Furthermore, we cultivated neuronal networks during 13 days with different doses of TNF-α in order to decipher its individual contribution among the other cytokines. Here we show that the ratio of tubulin to synapsin area was significantly higher in neuronal networks treated with either 4 or 2 doses of TNF-α, suggesting that TNF-α can promote the tubulin polymerization process. Assuming that TNF-α can bind to either TNFR1 or TNFR2 receptors, which lead respectively to the cell survival or the cell apoptosis, we studied the modulation of the both TNF-α receptors in response to the medium of mechanically activated astrocytes and different doses of TNF-α. Our findings indicate that the amount of both receptors increases with the maturation of the network. In addition, we observed a significant modulation of the amount of TNFR1 and TNFR2 in response to the media of injured astrocytes that leads to a large imbalance between both receptors, suggesting an important role for TNFα-signaling in the physiopathological mechanisms of TBI.

scholarly journals (79) Direct Bulblet Regeneration from Mature Embryo: A Rapid, Efficient and Genotype-independent In Vitro Morphogenesis Pathway for Preservation of Endangered Wild Populations of Fritillaria imperialis and Fritillaria persica

HortScience ◽  
2006 ◽  
Vol 41 (4) ◽  
pp. 1066C-1066
Author(s):  
Manijeh Mohammadi-Dehcheshmeh ◽  
Ahmad Khalighi ◽  
Esmaeil Ebrahimie ◽  
Manoochehr Sardari ◽  
Rohangiz Naderi
Keyword(s):  
Tissue Culture ◽  
Mature Embryo ◽  
Direct Organogenesis ◽  
Wild Populations ◽  
In Vitro Morphogenesis ◽  
Culture Techniques ◽  
Mature Embryos ◽  
Best Response ◽  
Heterozygous Plant

Wild populations of Fritillaria sp. have dramatically decreased in Iran because of pest overflow and continual grazing. Previous studies have shown that Fritillaria cannot rapidly and efficiently propagate by traditional methods. In vitro tissue culture techniques have shown high potential for micropropagation of endangered plants. The use of bulb-scale pieces for tissue culture can result in the destruction of the endangered parent plant. Fritillaria is a heterozygous plant in which the genetic content of each embryo is different from others, even on the same plant. In this study, mature embryos of F. imperialis and F. persica were used as explant for the first time. Embryos were extracted from seeds and cultured on B5 medium supplemented with various combinations of BAP (0, 0.1, 1 mg/L), NAA (0, 0.4, 4 mg/L), and IAA (0, 0.4, 4 mg/L). Embryo explant showed low genotype dependency between different heterogenous and heterozygote populations of both F. imperialis and F. persica. The best response of bulblet regeneration in both F. imperialis and F. persica was obtained from 1 mg/L BAP + 0.4 mg/L NAA+4 mg/L IAA and direct organogenesis pathway, with 15 bulblets per explant for F. imperialia and 20 for F. persica. Because of the large number of embryos in a plant and their different genetic contents, established in vitro propagation by using embryo explant in this study can provide broad genetic resources and variations. As explained above, in vitro protocols can play a major role in rescuing F. imperialis and F. persica from extinction.

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