Objective To improve the fixation method of the transmission electron microscope for better morphological preservation of mitochondria and lipid droplets in mouse brown adipose tissue.Methods The fixation method for mouse brown adipose tissue was optimized,mainly including an increased concentration of paraformaldehyde from 2％to 4％in the pre-fixative,employment of transcardial perfusion followed by immersion fixation in pre-fixation,and using imidazole-buffered osmium tetroxide as the post-fixative.The ultrastructures of brown adipocytes prepared by the improved method were observed and compared with those of a known standard protocol(3 mice in each group).The improved method was further validated in the quantitative analysis of mitochondrial cristae density and lipid droplets.Results The mitochondrial cristae and membrane structure of other organelles of brown adipocytes were better preserved using the optimized method compared with those of the standard method.Lipid droplets were presented as round structures with high electron density instead of vacuolated appearances.Using this method,we observed that the density of mitochondrial cristae and the content of lipid droplets increased in brown adipocytes after cold adaptation.Conclusion The optimized method can better preserve the ultrastructure of organelles in brown adipocytes,especially mitochondria and lipid droplets,and may be applicable for studying the ultrastructures remodeling of brown adipose tissue under different physiological or pathological conditions.
Objective To explore the protective effect of fosinopril(Fos)on streptozotocin(STZ)induced diabetic retinopathy(DR)mice and on the expression of angiotensin converting enzyme 2(ACE2)and vascular endothelial growth factor(VEGF)in DR mice.Methods Totally forty-eight healthy male Kunming mice,thirty-six were randomly selected,and a diabetic mouse model induced by STZ was constructed after 6 weeks of high-fat diet.After the successful establishment of the model,the thirty-six mice were randomly divided into three groups:model group,Fos low concentration(5 mg/kg)group,and Fos high concentration(10 mg/kg)group.The remaining twelve mice were served as the control group.After 8 weeks administration,the body weight and blood glucose level of mice in each group were determined.The change in the retinal structure was verified by HE staining.The serum level of VEGF was measured by ELISA.The expression of ACE2 in the retina tissue was verified by immunohistochemistry.The expression of ACE2 mRNA in diabetic retinopathy was analyzed by Real-time PCR.The levels of ACE2 and VEGF protein in diabetic retinopathy were detected by Western blotting.Results Fos(5 mg/kg,10 mg/kg)reduced significantly the proliferation of neovascularization in the inner boundary layer,down-regulated the expression of VEGF in the serum of diabetic mice and promoted the expression of ACE2 in the retina tissue of diabetic mice.In addition,the effect of the high concentration group of Fos had a stronger effect than the lower concentration group(P＜0.05).Conclusion Fos has a protective effect on the retinopathy of diabetic mice.This protective effect may be mediated through the increased expression of ACE2 and the reduction of VEGF expression.
Objective To explore the effect of melatonin(MLT)on the initiation of puberty in female mice and on the expression level of phosphatidylinositol-3-kinases(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway in the hypothalamus.Methods Seventy-eight 20-day-old female KM mice were randomly divided into melatonin(MLT)group and normal saline(NS)group,with 39 mice in each group.Starting at 22 days of age,the MLT group was given a subcutaneous injection of 1 mg/kg melatonin and the NS group was given an equal volume of saline.Thirty-two days of age were selected as the sampling point before puberty initiation and 13 mice were executed in each of the two groups,while 37 and 42 days of age were selected as the sampling point after puberty initiation and 13 mice were executed in each of the two groups.Observation of vaginal opening time in mice,weighing of ovaries and uterus to calculate organ indices.HE staining to observe the number of ovarian corpora lutea.The levels of serum luteinizing hormone(LH)were determined by ELISA.The mRNA and protein expression levels of PI3K/Akt/mTOR pathway in hypothalamus were detected by Real-time PCR and Western blotting.Results Compared with the normal saline group,mice in the melatonin group had significantly delayed vaginal opening time(P＜0.05),decreased significantly ovarian and uterine volume and index(P＜0.05),decreased significantly serum LH levels(P＜0.05),and decreased significantly mRNA and protein expression levels of the hypothalamic PI3K/Akt/mTOR pathway(P＜0.05).Conclusion Melatonin delays puberty initiation in mice by a mechanism that may be related to inhibition of the hypothalamic PI3K/Akt/mTOR signalling pathway.
Objective To study the role of complement C3a receptor(C3aR)in cognitive impairment in rats with sepsis induced by cecal ligation and puncture(CLP),and to explore the possible mechanism.Methods Totally 132 rats were randomly divided into sham operation group(sham group)and sepsis group(CLP group).The initial number of each group was 66 animals,and 22 animals were set at each time point.The SD rat CLP animal model was constructed,and serum and brain(hippocampus and prefrontal cortex)samples were collected at day 1,day 15 and day 30 after operation.The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and IL-6 in the samples were determined by ELISA.Western blotting detected Tau protein(Tau),phosphorylated Tau(p-Tau)and C3aR expression in brain samples.Totally 66 rats were randomly divided into 3 groups,sham operation group,CLP group and C3aR antagonist(C3aRa)intervention group.On 15th,17th,and 19th days after CLP,C3aRa intervened in rats,and they received inhibition avoidance test and object recognition test 30 days after CLP.The expressions of C3aR,Ionized calcium binding adapter molecule 1(Iba1),GFAP and p-Tau in the hippocampus were detected by immunofluorescence.Results Compared with the sham group,the serum and brain tissue(TNFα,IL-1β and IL-6)of rats in the CLP group first increased and then decreased within 30 days after CLP.In the hippocampus and prefrontal cortex of CLP group,Tau phosphorylation was significantly enhanced at day 30 and day 1 and 15 after surgery,respectively(P＜0.05).Compared with the sham group,C3aR protein levels in hippocampus and prefrontal cortex of rats in CLP group increased significantly at day 15 and 30 after operation(P＜0.05).Compared with the CLP group,the endogenous C3aR content decreased significantly after C3aRa intervention(P＜0.05),and Iba1,GFAP and p-Tau immunostaining were significantly inhibited(P＜0.05).The C3aRa intervention in CLP rats reversed the cognitive impairment.Conclusion C3aR plays an important role in the development of biochemical and behavioral changes commonly associated with the onset of sepsis-induced neurodegenerative processes.C3aRa can be injected into the hippocampus to counteract the neurodegenerative process induced by sepsis.
Objective To analysis risk factor and to construct a line graph prediction model for bone cement leakage after percutaneous transluminal vertebroplasty treatment in patients with osteoporotic spinal compression fractures.Methods A total of 236 patients with osteoporotic spinal compression fractures who came to our hospital from December 2019 to December 2021 were selected for the study,and they were divided into a leakage group(n=58)and a non-leakage group(n=178)according to whether bone cement leakage occurred after percutaneous transluminal vertebroplasty treatment.The clinical data were collected to analyze the factors associated with bone cement leakage;The work receiver operating characteristic(ROC)curves of the subjects were drawn to analyze the predictive value of each relevant factor on bone cement leakage;The Logistic multiple regression model was used to analyze the risk factors affecting bone cement leakage;The R language software 4.0"rms"package was used to construct the prediction model of column line diagram.Results The differences in age,bone density,degree of vertebral compression,vertebral endplate/posterior wall integrity,bone cement viscosity,and bone cement injection volume between patients in the leaky and non-leaky groups were statistically significant(P＜0.05).The area under curve(AUCs)for age,bone density,and cement injection volume to predict cement leakage were 0.804,0.825,and 0.803,respectively;The best cutoff values were 71 years,0.67 g/cm2,and 4.4 ml,respectively.Age(＞71 years),bone density(≤0.67 g/cm2),vertebral compression(severe),vertebral endplate/posterior wall integrity(no),cement viscosity(low viscosity),and bone cement injection volume(＞4.4 ml)were independent risk factors for bone cement leakage.The column line graph model predicted a C-index of 0.802(95％CI,0.689-0.868)for cement leakage,with a threshold＞0.19,and the column line graph model provided a net clinical benefit.Conclusion Age,bone density,degree of vertebral compression,vertebral endplate/posterior wall integrity,cement viscosity,and cement injection volume are independent risk factors for cement leakage,and the column line graph prediction model constructed with these predictors is of clinical application.
