Objective To investigate the influence of primary cultured neonatal rat hippocampal neurons caused by human cytomegalovirus (HCMV AD169) infection on intracellular calcium and its mechanism.Methods Twenty SPF SD rats born within 24 hours(10 cases of male and 10 cases of female) were assigned to establish the primary rat hippocampal neuronal monolayer cells; After cultured 8 days in vitro,the eligible cells were randomly divided into HCMV infection group,HCMV + MK-801 group,MK-801 group and control group,with 10 wells in each group.The fluorescence intensity values of the intracellular free calcium were detected after 24 hours of treatment with Fluo-3AM fluorescence staining.Results Inoculation of HCMV neurons after 24 h turned to round and swollen gradually,and 4days later,most of the cells disappeared; by immunohistochemistry in cultures of hippocampal neurons in HCMV,visible early proteins,brownish yellow granules,hematoxylin were found after being stained with brown pigment.The fluorescence intensity values of neuronal intracellular calcium (215.5 ± 14.9) in HCMV group was higher than that of control group (116.4 ± 5.9) (t =15.2,P ＜ 0.01),whilerise,that in MK-801 group (88.1 ± 4.5) was significantly lower than that of control group,with decreased rate of (24.0 ± 6.7) ％ (t =-9.3,P ＜ 0.01).The fluorescence intensity values of neuronal intracellular calcium in HCMV + MK-801 group (135.5 ± 8.6) was significantly decreased compared with that of HCMV group (215.5 ± 14.9),with decreased rate of (37.0 ± 3.4) ％ (t =11.3,P ＜ 0.01).Conclusions Intracellular calcium overload of cultured rat hippocampal neurons in vitro with HCMV AD16 strains infection can be detected.One of its main mechanisms is the N-methyl-D-aspartic acid receptor channel-mediated calcium influx.

Objective To establish and evaluate a reliable and highly reproducible neonatal rat model of hyper-bilirubinemia and to provide an experimental basis for research of kernicterus and related mechanism of neuroinjury.Meth-ods Sixty 7-day old SD rats (28 male and 32 female) were used in this study.Three doses of phenylhydrazine hydrochlo-ride (25, 50, and 75 mg/kg) were intraperitoneally injected respectively to the neonatal rats to establish models of hyper-bilirubinemia induced by hemolysis.The control group was set up at the same time.48 hours after the experimental treat-ment, the bilirubin in blood and brain tissue, neuron-specific enolase (NSE) of brain tissue, and hemoglobin were detec-ted to evaluate the models.Results Compared with the control group, the bilirubin in the blood and brain tissue and the brain tissue NSE in the three experimental groups were significantly higher than that in the control group (P<0.05), while hemoglobin content was significantly lower than that in the control group (P<0.05).The bilirubin of blood and brain tis-sue and brain tissue NSE in the 50 mg/kg and 75 mg/kg dose phenylhydrazine hydrochloride groups were significantly high-er than that of the 25 mg/kg dose group ( P<0.05) , while hemoglobin content was significantly lower than that of the 25 mg/kg dose group ( P<0.05 ) .There were no significant differences between the 50 mg and 75 mg dose groups ( P>0.05).Conclusions Intraperitoneal injection of phenylhydrazine hydrochloride can be used to produce neonatal rat mod-els of hyperbilirubinemia, mimicking the clinical features of this disease, and 50 mg/kg of phenylhydrazine hydrochloride is the best concentration.It is an ideal method to establish newborn rat models of hyperbilirubinemia.

Objective To establish the newborn rhesus monkey model of hemolytic hyperbilirnbinemia and provide an experimental basic model for research of hyperbilirubinemia.Methods Sixteen 3-day old newborn rhesus monkeys were divided into experimental group and control group,with 8 newborn rhesus monkeys in each group.Eight newborn rhesus monkeys in experimental group were treated with intravenous injection of l0 g/L phenylhydrazine hydrochloride (50 mg/kg) to establish model of homolytic hyperbilirubinemia.The newborn rhesus monkeys in control group were treated with intravenous injection of 9 g/L saline at the same time.Twenty-four hours and 48 hours after the experimental treatment,the bilirubin in blood was detected to evaluate the models,and the clinical manifestations of newborn rhesus monkeys with hyperbilirubinemia were recorded by using monitoring equipment.The brain slices were made to evaluate the model in 1 dead monkeys of experimental group.Results The newborn rhesus monkey of experimental group showed obvious skin,sclera jaundice and hemoglobinuria.The serum total bilirubin [(252.76 ± 63.42) μmol/L],unconjugated bilirubin[(165.85 ±44.93) pmol/L] and conjugated bilirubin [(87.16 ±21.22) μmol/L] in the experimental group were significantly higher than those [(20.62 ± 5.72) μmol/L,(7.93 ± 2.31) μmol/L,(12.51 ± 3.53) μmol/L] in the control group,and the differences were statistically significant (t =14.581,13.881,14.040,all P ＜ 0.01).The level of hemoglobin [(47.18 ± 10.09) μmol/L] in the experimental group was significantly lower than that of the control group [(136.85 ± 13.48) μmol/L],and the difference was statistically significant (t =-21.308,P ＜ 0.01).The results of pathological showed brain edema,rupture and eosinophilic and bilirubin deposition in the basal nuclei,and necrosis appeared in some severe parts.And there were different degrees of retardation and coordination disorders in the experimental group(s) newborn rhesus monkeys,but gradually returned to normal in 4 months later.Conclusion Intravenous injection of phenylhydrazine hydrochloride can be used to produce newborn rhesus monkey models of hemolytic hyperbilirubinemia.

Objective To study the effects of rehabilitation(RT)on cardiac function in cerebral infarct (CIF)patients with cardiac insufficiency(CIS).Methods Fifty-nine CIF patients with CIS were randomly divid- ed into a treatment group(T group,n=29)and a control group(n=30),and all patients were treated with routine pharmacotherapy for 2 months.In addition,RT was administrated in the T group at the same time.The grading of the New York Heart Association(NYHA)and the changes in cardiac function associated index were observed in both groups before and after treatment.Results Compared with the control group,NYHA grades,left ventricle ejection fraction(LVEF),the levels of brain natriuretic peptide(BNP)in the blood plasma,and the 6min walking range of the T group patients were all significantly improved after treatment(P＜0.05).Conclusion RT can improve car- diac function in CIF patients with CIS.

Objective To evaluate the effect of sedation with midazolam combined with propofol on delirium in mechanically ventilated patients in the intensive care unit (ICU).Methods Five hundred and twenty-two patients who required sedation and analgesia,endotracheal intubation and mechanical ventilation used to assist respiration,aged 28-64 yr,weighing 41-82 kg,were randomized into 2 groups according to the sedation protocols during therapy:sedation with midazolam group (group M,n =240) and sedation with midazolam + propofol group (group MP,n=232).In M and MP groups,sedation was induced with midazolam infusion 0.03-0.17 mg/min,and analgesia was induced with sufentanil infusion 0.07-0.14 μg/min.In group MP,when hemodynamics was stable,pressure support was 8-10 cmH2O,tidal volume＞400 ml,RR ＜25 bpm,and FiO2＜45％,sedation was induced with propofol infusion 0.8-2.0 mg/min instead,lasting for 12-24 h.Richmond Agitation Sedation Scale score was maintained at-1 to-2 during vcntilation.The development and duration of delirium were recorded.Delirium was divided into hyperactive delirium,hypoactive delirium and mixed delirium 3 subtypes,and the development and duration of the 3 subtypes of delirium were also recorded.Results There was no significant difference between the two groups in the incidence and duration of delirium.Compared to group M,the incidence of hyperactive delirium was significantly decreased,and no significant change was found in the incidence of hypoactive delirium and mixed delirium and the duration of the 3 subtypes of delirium in group MP.Conclusion Sedation with midazolam and propofol can decrease the development of hyperactive delirium,but can not shorten the duration of delirium in mechanically ventilated patients in the ICU.

OBJECTIVETo investigate the effect of glycosylation at Asn302 of pro-urokinase (pro-UK) on the stability in culture supernatant.

METHODSNonglycosylated pro-UK was constructed by site-directed mutagenesis of Asn302 to Ala302. The pro-UK mutant and native pro-UK were transfected into dhfr(-)-CHO cells, and serum-free culture supernatant was harvested and incubated at 4 degrees C and 37 degrees C, respectively. The pro-UK activity in culture supernatant was measured by the optical density (OD) increase with time (12 hours) at 405 nm. Without thermolysin activation, the percentage of single chain pro-UK was measured.

RESULTSAfter 48 hours of incubation at 4 degrees C, the activities of pro-UK mutant and native pro-UK decreased 3.7% and 2.9% respectively, and at 37 degrees C decreased 37.9% and 23.5%, respectively. The total activity of native pro-UK was significantly higher than that of nonglycosylated mutant at 37 degrees C. The single-chain percentage of native pro-UK was higher than that of nonglycosylated mutant at both 4 degrees C and 37 degrees C.

CONCLUSIONHigher temperature increases the proteolysis of pro-UK. The glycosylation site on Asn302 is beneficial to pro-UK stability in culture supernatant.

Amino Acid Substitution ; Animals ; Asparagine ; chemistry ; Base Sequence ; CHO Cells ; Cricetinae ; Cricetulus ; Culture Media ; DNA Primers ; genetics ; Enzyme Stability ; Glycosylation ; Humans ; In Vitro Techniques ; Mutagenesis, Site-Directed ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Urokinase-Type Plasminogen Activator ; chemistry ; genetics ; metabolism

AIMThe relationship between gastric acid secretion and ATP level, and regulation of uncoupling protein 2 (UCP2) mRNA expression by vagus nerve were studied in vagotomies rats.

METHODSWith the high selective vagotomy model, the gastric acidity was titrated to pH 7.0 with 0.1 mol/L NaOH solution and ATP contents were quantified by using fluorimetry. The expression of UCP2 mRNA was observed by using Northern blot in stomach of rats.

RESULTSBoth of gastric acidity and ATP contents in stomach body decreased significantly at 24 h after vagotomy. The expression of UCP2 mRNA was markedly increased as compared with sham operation group.

CONCLUSIONATP contents decreased and vagus nerve down-regulates expression of UCP2 mRNA in stomach corpus in vagotomies rats. The results indicates that vagus nerve could underlay the gastric acidity by inhibiting expression of UCP2 mRNA and increasing ATP contents in rats.

Adenosine Triphosphate ; metabolism ; Animals ; Gastric Acid ; secretion ; Ion Channels ; genetics ; metabolism ; Male ; Mitochondrial Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Uncoupling Protein 2 ; Vagotomy ; Vagus Nerve ; metabolism

Objective To investigate the incidence of hearing disorder and analyse the high-risk factors with hearing injury in newborns with hyperbilirubinemia.Methods The newborns with hyperbilirubinemia who admitted to the department of neonate,were received the distortion product otoacoustic emission(DPOAE)test when they recovered from hyperbilirubinemia;those babies who didn′t pass the first test received screening again in 42 days after birth.Those babies who didn′t pass the second test received auditory brain stem response(ABR)test.Results Fifty-eight(33.2%)newborns didn′t pass the first DPOAE test among 235 newborns with hyperbilirubinemia;11(18.9%)infants didn′t pass the second DPOAE test among 58 infants;5 infants failed to pass the ABR test,the ratio of hea-ring disorder in newborns with hyperbilirubinemia was 2.13%;18(9.9%)newborns didn′t pass the first DPOAE test among 182 normal newborns,and those infants all passed the second DPOAE test.Conclusions Hyperbilirubinemia is high-risk population of hearing disorder.The congenital cytomegalovirus infection,neonatal septicemia and hemolytic disease of newborn are the high risk factors responsible for hearing disorder.All high risk newborns should recieve hearing examination regularly.

OBJECTIVETo investigate the clinical outcomes of semicircular decompression in treating old thoracolumbar fractures and intractable neuropathic pain.

METHODSFrom September 2009 to September 2013, 21 patients with old thoracolumbar fracture and intractable neuropathic pain were treated with semicircular decompression. Among initial surgery, posterior pedicle screw fixation was used in these patients, with or without laminectomy. All patients were male, range in age from 20 to 28 years old with an average of (25.00±2.38) years. Vertebral body residual bone block resulted in intra-spinal placeholder more than 50%. All patients were complete spinal cord injury (ASIA grade) or cauda equina injury. VAS scores was from 6 to 10 points with the mean of 7.14±0.91. In these patients, MRI, CT, X-rays were performed; denomination and dosage of analgesics were recorded; nerve function and pain status were respectively evaluated by ASIA grade and VAS score before and after operation.

RESULTSAll patients were followed up from 8 to 32 months with an average of (17.29±6.02) months. All bone fragments of spinal canal were removed and spinal cord decompressions were achieved. At final follow-up, VAS scores were from 0 to 8 points with an average of (2.43±2.46) points, and were obviously reduced than peroperative data (P<0.05). Eleven cases of them stopped analgesic intake and 7 cases reduced using. Three patients' symptoms and VAS scores were not improved.

CONCLUSIONOld thoracolumbar fractures and intractable neuropathic pain need receive imaging examination as soon as possible and consider semicircular decompression therapy if bone fragments were in vertebral canal and spinal canal stenosis existed. This therapy can effectively relieve pain and profit nerve functional recovery.

Adult ; Decompression, Surgical ; methods ; Humans ; Lumbar Vertebrae ; injuries ; surgery ; Male ; Neuralgia ; etiology ; surgery ; Pain, Intractable ; etiology ; surgery ; Spinal Fractures ; physiopathology ; surgery ; Thoracic Vertebrae ; injuries ; surgery ; Visual Analog Scale ; Young Adult

OBJECTIVETupaia belangeri (tree shrew) has a close phylogenetic relationship with primates and has been shown to be susceptible to a variety of human viruses. This study was conducted to investigate whether or not hepatitis C virus (HCV) could infect primary tupaia hepatocytes (PTHs) in vitro.

METHODSSerum-derived HCV was cultivated with PTHs, and then positive and negative strand HCV RNA in PTHs, as well as the encapsidated HCV RNA in the culture medium were detected to evaluate the infection. Virus from the culture medium of the infected PTHs was passed to naïve PTHs, and the quasispecies of HCV were compared among the inoculum and PTHs after infection and passage.

RESULTSBoth positive and negative strand HCV RNA were detected in PTHs after infection. The negative strand RNA was detectable from day 5 to day 10 after infection, while the positive strand RNA was positive up to day 14. HCV RNA, which was RNase resistant, could be detected from the culture medium of the infected PTHs from day 3 to day 14. Production of infectious virons of PTH were demonstrated by passage HCV to naïve PTHs. Compared analysis of HCV quasispecies after infection and passage showed that PTHs were selectively infected with defined HCV quasispecies, and new quasispecies emerged in PTHs after passage.

CONCLUSIONThe present study strongly indicates that PTHs could be infected by HCV and support HCV replication in vitro. Our results would be helpful for the establishment of a tupaia model of HCV infection.

Animals ; Cells, Cultured ; Hepacivirus ; pathogenicity ; physiology ; Hepatocytes ; virology ; Tupaia ; Virus Replication