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OBJECTIVE：To study the effects of virus in activation treatment of plasma specimen on plasma concentration determination of voriconzole ，linezolid，vancomycin and teicoplanin. METHODS ：The remaining plasma of 36 inpatients in our hospital after routine blood concentration examination of voriconazole ，linezolid，vancomycin and teicoplanin were collected as specimen（9 drug-contained plasma specimens for each drug ），and merged into three different concentration levels （low，medium， high）of mixed samples according the results of routine blood test. Then the mixed samples with different concentration levels were divided into inactivated group and non-inactivated group ，with 3 samples in each group. The inactivated plasma samples were heated at 56 ℃ for 30 min in metal bath with constant temperature. Non-inactivated group were not treated. After pretreating plasma sample of 2 groups，2-dimensional liquid chromatography was used to detect plasma concentration of the four drugs ；the difference of detection result between inactivated group and non-inactivated group were analyzed. RESULTS ：Plasma samples containing voriconazole，linezolid，vancomycin and teicoplanin were still stable after heating at 56 ℃ for 30 min in metal bath with constant temperature. Compared with non-inactivated group ，relative error of plasma concentration detection result of above 4 drugs were all lower than 15％ in low ，medium，high concentration mixed samples of inactivated group. CONCLUSIONS ：Plasma samples can be inactivated by heating at 56 ℃ for 30 min in metal bath with constant temperature ，when the plasma concentration of voriconazole，linezolid，vancomycin and teicoplanin are determined by 2-dimensional liquid chromatography.
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Objective To observe diffusion changes of epiphysis of femoral head with ischemia of difference phases by line-scan diffusion weighted imaging(LSDWI),and determine whether LSDWI can provide temporal information and severity about ischemia of epiphysis.Methods lschemia was surgically induced in one hip of each piglet(n=25)and the other hip served as a normal control.Piglets were imaged before surgery and at 3 hours,72 hours and 1,3 and 6 weeks after surgery by using LSDWI.Apparent difrusion coefficients(A DC)in epiphysis of the femoral heads were calculated.Significant difierences in ADC values between ischemia group and control group were found by using paired t-test.After scan at individual time points,5 piglets were sacrificed for histological study each time.Results The ADC value in the ischemic femoral heads f(1.22±0.37)×10-3 mm2/s]decreased significantiy at 3 hours after surgery (t=3.914,P＜0.01),compared to that in control[(1.73±0.33)×10-3mm2/s},and increased at 72 hours[(2.15±0.32)×10-3mm2/s versus(1.70±0.22)×10-3 mm2/s](t=3.348,P＜0.01).Then ADC valne kept increasing until 6 weeks after surgery[(1.61±0.27)×10-3mm2/s in ischemia side vs (1.11±0.45)×10-3mm2/s in the control](t=4.136,P＜0.01).rrhe percentage change of the ADC value significandy increased at 3 hours,72 hours,1 and 3 week(s)after the surgery(P＜0.01),compared to that at the prior neighboring time point.No significant increase in the percentage change of ADC value was found between the 3rd week and the 6th week after the surgery(t=2.29.P＞0.05).Histological examinations revealed abnormal thickening within epiphyseal cartilage,and cartilaginous islands within ossified tissues.Growth disturbante wag found in form of focal growth plate disruption.Conclusions Dynamic changes of ADC values were found with the prolonged ischemia of the femoral head by LSDWI.It could serve as a useful marker for evaluating duration and extent of ischemic epiphyseal disruption.
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To examine the aging-related changes of microglia and astrocytes in hypothalamus of rats after intraperitoneal injection of hypertonic saline in rats, old- and young-aged rats were injected with hypertonic saline solution into peritoneal cavity. Lectin histochemical techniques using Ricinus communis agglutinin-1 (RCA-1) and immunocytochemical method employing antibody against glial fibrillary acidic protein (GFAP) were used to demonstrate microglia and astrocytes in the hypothalamus of the rats, and the positively-stained cells were analyzed by computer-assisted image analysis system. Our results showed that the numbers of microglia and astrocytes were significantly increased in the hypothalamus of old-aged rats. After intraperitoneal injection of hypertonic saline, the number of microglia was significantly decreased in the hypothalamus of both young- and old-aged groups. After introperitoneal injection of hypertonic saline, the number of GFAP positive cells was significantly increased in the hypothalamus of young rats, but the number of GFAP positive cells did not show significant change in the hypothalamus of old rats. It is concluded that in the hypothalamus of old-aged rats, the increase of microglia may be related with the aging or degeneration of neurons, and the increase of astrocytes may provide more nourishment required by the aged neurons. The microglia and astrocytes in the hypothalamus of the two group rats may be affected by hypertonic saline, and the response of these cells to the stimuli is characterized by some aging-related changes.
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AIM: To probe into tau hyperphosphorylation at PHF- 1 sites induced by glycogen synthase kinase - 3β(GSK- 3β) in vivo. METHODS: Twenty - one rats were randomly allocated to three groups as follows: GSK - 3β transfection group, vector group and control group; 0.1 μg/3μL GSK- 3β- HA plasmid or vector was injected bilaterally into cerebrum of the rats respectively, rats without injection were controls. Western blotting and immunohistochemical staining of cortex were carried out to detect the expression of GSK- 3β- HA plasmid and tau phosphorylation using phosphorylation- dependent tau antibody PHF- 1. RESULTS: After transfection with GSK- 3β- HA for 48 h, GSK - 3β - HA was expressed in GSK- 3β transfection group; and hyperphosphorylated tau at PHF- 1 sites accumulated in neurons in the transfected areas. The hyperphosphorylated tau colocalized largely with GSK- 3β expressed by the transfected GSK- 3β plasmid. CONCLUSIONS: Transfection with GSK- 3βin vivo can induce tau hyperphosphorylation involving the pathogenesis of neurodegenerative disorders. These data further prove that GSK- 3β is a key kinase to induce tau hyperphosphorylation and may be a therapeutic target for tauopathy- related neurodegenerative diseases.
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To examine the aging-related changes of microglia and astrocytes in hypothalamus of rats after intraperitoneal injection of hypertonic saline in rats, old- and young-aged rats were injected with hypertonic saline solution into peritoneal cavity. Lectin histochemical techniques using Ricinus communis agglutinin-1 (RCA-1) and immunocytochemical method employing antibody against glial fibrillary acidic protein (GFAP) were used to demonstrate microglia and astrocytes in the hypothalamus of the rats, and the positively-stained cells were analyzed by computer-assisted image analysis system. Our results showed that the numbers of microglia and astrocytes were significantly increased in the hypothalamus of old-aged rats. After intraperitoneal injection of hypertonic saline,the number of microglia was significantly decreased in the hypothalamus of both young- and oldaged groups. After introperitoneal injection of hypertonic saline, the number of GFAP positive cells was significantly increased in the hypothalamus of young rats, but the number of GFAP positive cells did not show significant change in the hypothalamus of old rats. It is concluded that in the hypothalamus of old-aged rats, the increase of microglia may be related with the aging or degeneration of neurons, and the increase of astrocytes may provide more nourishment required by the aged neurons. The microglia and astrocytes in the hypothalamus of the two group rats may be affected by hypertonic saline, and the response of these cells to the stimuli is characterized by some aging-related changes.
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To study the prevention of dauricine (Dau) on bradykinin (BK) induced alteration of intracellular calcium homeostasis and tau phosphorylation, fluorescence spectrophotometer with dual excitation was utilized to measure the intracellular calcium concentration ([Ca2+]i), MTT to detect cell viability and immuncytochemistry to examine tau phosphorylation. The results showed (1) cells treated with BK 1 μmol/L induced a transit increase in [Ca2+]i in all the cell lines detected, among them, the sustained increase of [Ca2+]i level was only seen in PS1Δ9/APPswe cell at 2 h and 24 h after the treatment. Dau (3μmol/L or 6 μmol/L) prevented BK-induced transit and sustained elevation and fluctuation of [Ca2+]i;(2) BK treatment decreased the cell metabolism detected at 2 h in PS1Δ9/APPswe and Dau antagonized the effect; (3) BK induces Alzheimer-like tau hyperphosphorylation at tau-1 epitope and Dau partially antagonized this effect. In conclusion,Dau inhibits BK-induced disturbance in intracellular calcium homeostasis and tau hyperphosphorylation at tau-1 sites.
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To explore the effect of different concentrations of corticosterone (CORT) on primary cultured hippocampal neurons and their Ca2+/CaMK II expression and possible mechanism, the changes of hippocampal neurons were observed in terms of morphology, activity of cells, cell death, concentrations of cytosolic free calcium, and the expression of CaMK II by using MTT assay, flow cytometry, fluorescent labeling of Fura-2/AM and Western blotting after 10(-7), 10(-6) and 10(-5) mol/L of CORT was added to culture medium, The evident effect of 10(-6) and 10(-5) mol/L of CORT on the morphology of hippocampal neuron was found. Compared with control neurons, the activity of the cells was markedly decreased and [Ca2+]i increased in the neurons treated with 10(-6) and 10(-5) mol/L of CORT, but no change was observed in the neuron treated with 10(-7) mol/L of CORT. The death was either by way of apoptosis or necrosis in the cells treated with 10(-6) and 10(-5) mol/L of CORT respectively. The correlation analysis showed that a reverse correlation existed between [Ca2+]i and the expression of CaMK II. Either apoptosis or necrosis occurs in the hippocampal neurons treated with CORT. The increased hippocampal [Ca2+]i is both the result of CORT impairing the hippocampal neurons and the cause of the apoptosis of hippocampal neurons and the decreased CaMK II expression.
Subject(s)
Animals , Rats , Apoptosis , Calcium , Metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calcium-Calmodulin-Dependent Protein Kinases , Genetics , Cells, Cultured , Corticosterone , Pharmacology , Hippocampus , Cell Biology , Neurons , Cell Biology , Rats, WistarABSTRACT
Objective To compare the survival of isolated chromaffin cells with that of adrenal medullary pieces implanted into the subarachnoid space .Methods Forty male Sprague Dawley rats, 180 220g, were randomly allocated to be implanted into the subarachnoid space with the homologous adrenal medullary pieces washed with PBS(group A,n=20) or the isolated chromaffin cells 5?107 in 10?l suspension (group B,n=20), respectively, after the implants were in vitro cultured in three days . The thermal threshold was determined before ,5 and 10 weeks after the transplantation. Five or ten weeks after the transplantation, 6 rats of either group were randomly selected to obtain the adrenal medullary grafts from the subarachnoid space or the sediments of cerebrospinal fluid ,observed with an electron microscopy.Results Five weeks after transplantation, the thermal threshold in both groups increased markedly,but without significant difference between them ,and there were intact chromaffin cells in the sections of both groups. Ten weeks after the operation, the thermal threshold in group A decreased to basaline, and was significantly lower than in group B; no intact chromaffin cells were found ,with the infiltration of a large number lymphocytes in the sections of group A , and in the sections of group B, the intact chromaffin cells were found with the infiltration of lymphocytes in lower amount and density. Conclusions The isolated chromaffin cells implantation is superior to the adrenal medulla pieces implantation for analgesia.
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AIM: To probe into tau hyperphosphorylation at PHF-1 sites induced by glycogen synthase kinase-3? (GSK-3?) in vivo. METHODS: Twenty-one rats were randomly allocated to three groups as follows: GSK-3? transfection group, vector group and control group; 0.1 ?g/3 ?L GSK-3?-HA plasmid or vector was injected bilaterally into cerebrum of the rats respectively, rats without injection were controls. Western blotting and immunohistochemical staining of cortex were carried out to detect the expression of GSK-3?-HA plasmid and tau phosphorylation using phosphorylation-dependent tau antibody PHF-1. RESULTS: After transfection with GSK-3?-HA for 48 h, GSK-3?-HA was expressed in GSK-3? transfection group; and hyperphosphorylated tau at PHF-1 sites accumulated in neurons in the transfected areas. The hyperphosphorylated tau colocalized largely with GSK-3? expressed by the transfected GSK-3? plasmid. CONCLUSIONS: Transfection with GSK-3? in vivo can induce tau hyperphosphorylation involving the pathogenesis of neurodegenerative disorders. These data further prove that GSK-3? is a key kinase to induce tau hyperphosphorylation and may be a therapeutic target for tauopathy-related neurodegenerative diseases.