Evaluation of direct intramural injection to the bladder wall as a method for developing orthotopic tumor models

Background:Bladder cancer poses a great burden on society and its high rate of recurrence and treatment failure necessitates use of appropriate animal models to study its pathogenesis and test novel treatments.Orthotopic models are superior to other types since they provide a normal microenvironment.Four methods are described for developing bladder cancer models inside the animal’s bladder.Direct intramural injection is one of these methods and is widely used.However,its efficacy in model development has not yet been studied.We aimed to evaluate the efficacy and success rate of the direct intramural injection method of developing an orthotopic model for the study of bladder cancer.Method:Tumor cell lines were prepared in four microtubes.Aliquots of 200×10^(3) cells were injected through a 27 gauge needle into the ventral wall of the bladders of 4male and 4 female BALB/c mice following a midline 1 cm laparotomy incision.In addition,1 million cells from each microtube were injected into the flanks of control mice.To prevent infection and alleviate pain,5 mg/kg enrofloxacin and 2.5 mg/kg flunixin meglumine,respectively,were injected subcutaneously.Results:Tumors formed in all mice,resulting in 100% take rate and zero post-operation mortality.Surgery time was≤15 min per mouse.In two mice,tumors were found in the peritoneal space as well.Conclusion:Direct intramural injection is a rapid,reliable,and reproducible method for developing orthotopic models of bladder cancer.It can be done on both male and female mice and only requires readily available surgical tools.However,needle track can result in cell spillage and peritoneal tumors.

Effects of vascular endothelial growth factor supplementation and alginate embedding on human oocyte maturation in vitro

Objective:To evaluate whether the use of vascular endothelial growth factor(VEGF)with alginate increases oocyte maturation following in vitro maturation.Methods:This experimental study was performed on 150 immature oocytes(germinal vesicle oocytes)from females who were candidates for assisted reproductive technology.The germinal vesicle oocytes were randomly placed in the control,alginate,and VEGF plus alginate groups.The basic culture medium for oocytes culture(tissue culture medium 199,follicle-stimulating hormone 0.075 IU/mL,and fetal bovine serum 10%)was used in the control,alginate,and VEGF plus alginate groups.For the treatment groups(alginate,and VEGF plus alginate groups),alginate(8%)and VEGF(5 ng/mL)were added to the basic culture medium.After culture,immature oocytes were considered as oocytes unchanged in the nucleus whereas oocytes with a polar body were considered as mature oocytes(metaphaseⅡstage).The mature oocytes in each group were fertilized by intracytoplasmic sperm injection and formed embryos were evaluated by reverse microscope.Results:The oocyte maturation rate(metaphaseⅡ)significantly(P<0.05)increased in the alginate plus VEGF group as compared with the alginate alone and control groups during in vitro maturation.On day 2,the cleavage rates were significantly different in the matured oocytes between the treatment groups and the control group.The percentage of the two-cell stage,four-cell stage and eight-cell embryos was significantly higher in the treatment groups compared with the control group(P<0.05).Conclusions:Supplementation of VEGF with alginate can improve oocyte maturation in culture media.VEGF with alginate may promote the quality of nuclear and cytoplasmic maturation of human oocytes in vitro.