Determination of the Early Time of Death by Computerized Image Analysis of DNA Degradation： Which Is the Best Quantitative Indicator of DNA Degradation？

This study evaluated the correlation between DNA degradation of the splenic lympho-cytes and the early time of death,examined the early time of death by computerized image analysis technique(CIAT) and identified the best parameter that quantitatively reflects the DNA degradation.The spleen tissues from 34 SD rats were collected,subjected to cell smearing every 2 h within the first 36 h after death,stained by Feulgen-Van's staining,three indices reflecting DNA content in splenic lymphocytes,including integral optical density(IOD),average optical density(AOD),aver-age gray scale(AG) were measured by the image analysis.Our results showed that IOD and AOD decreased and AG increased over time within the first 36 h.A stepwise linear regression analysis showed that only AG was fitted.A correlation between the postmortem interval(PMI) and AG was identified and the corresponding regression equation was obtained.Our study suggests that CIAT is a useful and promising tool for the estimation of early PMI with good objectivity and reproducibility,and AG is a more effective and better quantitative indicator for the estimation of PMI within the first 36 h after death in rats.     

Image Analysis for Degradation of DNA in Retinal Nuclei of Rat after Death

The changes of retinal nuclear DNA content in rats after death was detected and the rela-tionship between degradation of retinal nuclear DNA and postmortem interval (PMI) was analyzed. Ninety healthy adult SD rats, female, weighing 250±10 g, were randomly divided into 15 groups. At 20 ℃, the retinal cells were withdrawn every 2 h within 0 to 28 h after death and stained with Feul-gen-Vans. Index of density (ID), integral absorbance (IA) and average absorbance (AA) in retinal nu-cleus were analyzed by image analysis system. And the obtained data were subjected to linear regres-sion analysis by using SPSS12.0 software. The results showed that in retinal nucleus, AA and IA were gradually declined with the prolongation of PMI, while ID had an increased tendency. Within 28 h after PMI, the regression equations were as follows: YAA=-0.009XAA 0.590 (R2=0.949), YIA=-0.097XIA 18.903 (R2=0.968), YID=0.122XID 2.246 (R2=0.951). It was concluded that retinal nuclear DNA after death in rats was degraded gradually and had a good correlation with PMI.     

Gap junctional protein connexin 43 in rat detrusor muscle with unstable bladder

Background Unstable bladder is one of the common clinical dysfunctions of the lower urinary tract. Gap junctions （GJs） are the plaques of aqueous channels that facilitate electrical and metabolic communication between the intracellular compartments of adjacent cells, exchange of nutrients and ions between connected cells and transfer of electrical signals In the present study we investigated the quantitative alterations of the GJ in the rat detrusor muscle and its functional changes related to the developing of unstable bladder （USB）. Methods Thirteen female Wistar rats （study group） with obstructive unstable bladder as determined by urodynamic study and 10 sham-operated rats （control group） were sacrificed at 6 weeks after surgery. Cystometric investigation, and the content and distribution of the GJ protein connexin 43 （Cx43） in the detrusors which were taken from the bladder of the rats were studied by Western blot and laser confocal microscopy with a double label immunohistochemistry technique. Results The expression of Cx43 was found adjacent to the detrusor with the laser confocal microscopy. The Cx43 expression increased markedly in the study group （pixel density 29.5±13.9, staining size （17.9±8.8） μm2） compared with the control group （pixel density 14.2±2.2, staining size （5.7±3.1） pm2, P 〈0.05）. Western blot analysis demonstrated that Cx43 in the study group （the average gray level was 31.066） was significantly higher than in the control group （the average gray level was 11.701, P 〈0.01 ）. Conclusion The increase of GJ leading to a intercellular excitatory communication is one of the important mechanisms related to developing unstable bladder.     

Bioluminescent assay of microbial ATP in postmortem tissues for the estimation of postmortem interval

To study the relationship between changes of microbial ATP in four kinds of murine tis-sues and the postmortem interval （PMI）, healthy SD rats were sacrificed and their muscles, livers, spleens and kidneys were sampled at different postmortem intervals. The concentration of microbial ATP was detected using bioluminescent assay and the data was statistically analyzed. The concentra-tion of microbial ATP in muscle increased with PMI time. The peak appeared at the 7th day after death, and at the 10th day, microbial ATP in muscle tissue increased again. In internal organs, the peaks of microbial ATP were observed at the 8th day after death and the level decreased during 8–10 d. The differences in microbial ATP concentration in liver, spleen and kidney were not statistically significant. During day 0 to day 9 after death, the correlation was best between PMI and microbial ATP in muscle. With PMI as the independent variable, the cubic polynomial regression equation was Y=0.02X3–0.166X2–0.666X＋13.412 （R2=0.989, P〈0.01）. In internal organs, the best correlation was found between PMI and microbial ATP during day 0 to day 10. With PMI as the independent variable, the cubic polynomial regression equation was Y=0.016X3–0.127X2–0.809X＋13.324 （R2=0.986, P〈0.01）. There existed high correlations between PMI and microbial ATP concentration in rat tissues. Since only a small amount of tissue was needed for the detection and the sample was not affected by self-decomposition, the method may extend the time range of PMI estimation.     

Outcome of kidney transplantation between controlled cardiac death and brain death donors: a meta-analysis

Background Our goal was to evaluate the outcomes of kidney transplants from controlled cardiac death donors compared with brain death donors by conducting a meta-analysis of cohort studies.Methods The PubMed database and EMBASE were searched from January 1980 to July 2013 to identify studies that met pre-stated inclusion criteria.Reference lists of retrieved articles were also reviewed.Two authors independently extracted information on the designs of the studies,the characteristics of the study participants,and outcome assessments.Results Nine cohort studies involving 84 398 participants were included in this meta-analysis; 3 014 received kidneys from controlled cardiac death donors and 80 684 from brain death donors.Warm ischemia time was significantly longer for the controlled cardiac death donor group.The incidence of delayed graft function was 2.74 times （P 〈0.001） greater in the controlled cardiac death donor group.The results are in favor of the brain death donor group on short-term patient and graft survival while this difference became nonsignificant at mid-term and long term.Sensitivity analysis yielded similar results.No evidence of publication bias was observed.Conclusion This meta-analysis of retrospective cohort studies suggests that the outcome after controlled cardiac death donors is comparable with that obtained using kidneys from brain death donors.     

The Regulatory Action of Radix Astragali on M-Cholinergic Receptor of the Brain of Senile Rats

The changes in density of M-cholinergic receptors in different areas of senile rats and the regulatoryaction of Huang Qi (黄芪 Radix Astragali,a drug for warming yang and replenishing qi) were observedby autoradiography.The results showed that the gray scale displayed in brain sections was clear andmainly distributed in the cortex,hippocampus and striate body,while that due to nonspecificcombination was negligible.The gray scale in the cortex,hippocampus and striate body of theexperimental group was markedly lower than that in the young control rats,decreased respectively by24.87%,14.12% and 12.76% (all P<0.05);but it was obviously higher than those in the senile controlrats,increased respectively by 24.15%,14.38% and 13.47% (P<0.05).The data indicate that Huang Qi(黄芪 Radix Astragali) may up-regulate the decreased density of M-cholinergic receptors in the brain ofsenile rats.     

Application of Proton Magnetic Resonance Spectroscopy and Computerized Tomography in the Diagnosis and Treatment of Nonalcoholic Fatty Liver Disease

In order to investigate the application of proton magnetic resonance spectroscopy （ill-MRS） and computerized tomography （CT） in the quantitative diagnosis of nonalcoholic fatty liver disease （NAFLD） and evaluation of therapeutic effects, 22 patients with NAFLD were selected according to the Chinese Medical Association＇s （CMA） standard of the NAFLD in comparison with 20 healthy volunteers （as control group）. Blood samples for biochemistry were collected. The severity of hepatosteatosis was evaluated by ^1H-MRS scan and CT scan of liver. The intrahepatic content of lipid （IHCL） and CT value ratio of liver to spleen were calculated. The patients in NAFLD group were treated with Ganzhixiao Capsule for 8 weeks. The changes in IHCL and CT value ratio of liver to spleen were observed before and after treatment. In NAFLD group serum ALT, TG, IHCL calculated by ^1HMRS were increased and CT value ratio of liver to spleen decreased significantly as compared with control group. After treatment for 8 weeks serum ALT, TG, IHCL were decreased significantly, while CT value ratio of liver to spleen increased significantly in NAFLD group. It was suggested that IHCL could be measured precisely by ^1HMRS. NAFLD was treated effectively by Ganzhixiao capsule.     

Expression of Arginyl-tRNA Synthetase in Rats with Focal Cerebral Ischemia

Aminoacyl-tRNA syntheses （AARS） can catalyze the adenosine triphosphate （ATP）-dependent acylation of their cognate tRNA（s） with a specific amino acid. They can be seen as an index to reflect the energy metabolic rate of ischemic brain cells in ischemic penumbra. This study ex- amined the relationship between arginyl-tRNA synthetase （ArgRS）, one of the AARS, and cerebral ischemia in rats. The model of middle cerebral artery occlusion （MCAO） was established in rats. The expression levels of ArgRS protein and mRNA were detected in rat brain tissues at different time points following MCAO by Western blotting and RT-PCR, respectively. The results showed that the MCAO model was successfully established. Western blotting and RT-PCR analysis revealed that the ArgRS protein and mRNA were expressed in brain cells in both ischemic and normal penumbra tissues. The expression levels of ArgRS protein and mRNA peaked at 6 h after MCAO and decreased gradually. At 24 h, the expression levels of ArgRs protein and mRNA in ischemic penumbral tissues were lower than those in normal tissues. The expression levels of ArgRS mRNA and protein in ischemic penumbra var- ied with ischemic time, suggesting that the energy metabolism of brain cells in penumbra changed dy- namically after ischemia to ensure the endogenous self-protection of the body. The brain oxygen supply should be improved as soon as possible, especially within 6-12 h after ischemia, so as to meet the de- mand for energy metabolism in ischemic penumbra and make sure the cell structure remains stable.     

Quantitative Study of Iron Metabolism-related Genes Expression in Rat

Objective To investigate the multiple iron metabolism-related genes expression, its regulation by iron and the expression correlation among the genes in rat tissues. Methods Two groups （n=30） of Sprague-Dawley female weanling rats were fed with a control diet and an iron deficient diet respectively for 4 weeks. All rats were then sacrificed, and blood and tissue samples were collected. The routine blood examination was performed with a veterinary automatic blood cell analyzer. Elemental iron levels in liver, spleen and serum were determined by atomic absorption spectrophotometry. The mRNA expression of genes was detected by real-time fluorescence quantitative PCR. Results After 4 weeks, the hemoglobin （Hb） level and red blood cell （RBC） count were significantly lower in the iron deficient group compared with those in the control group. The iron levels in liver, spleen and serum in the iron deficient group were significantly lower than those in the control group. In reference to small intestine, the relative expression of each iron-related gene varied in the different tissues. Under the iron deficiency, the expression of these genes changed in a tissue-specific manner. The expression of most of the genes significantly correlated in intestine, spleen and lung, but few correlated in liver, heart and kidney. Conclusion Findings from our study provides new regulation by iron and correlation among the mRNA divalent metal transporter 1, ferritin, iron regulation protein, hepcidin, ferroportin 1 and hephaestin in intesti understandings about the relative expression, expressions of transferrin receptors 1 and 2, proteins 1 and 2, hereditary hemochromatosis ne, liver, spleen, kidney, heart, and lung of rat.     

死后5～36 h人脑细胞DNA降解含量的图像分析

目的通过对死后5～36 h脑细胞降解后残存DNA含量的定量分析来推断其早期死亡时间.方法选取32例已知死亡时间的人体脑组织,在死后5～36 h内每小时进行细胞学涂片、Feulgen-Vans染色,采用图像分析技术检测反映脑细胞核DNA含量的平均光密度、积分光密度、平均灰度等灰度参数,分析脑细胞残存DNA的含量与其对应的死亡时间的变化规律.结果在5～36 h内平均光密度、积分光密度随死亡时间的延长而逐渐减小、平均灰度逐渐增大,且其均值均与死亡时间显著性相关,并得出对应的回归方程.结论反映人脑细胞DNA含量改变的灰度参数均与死亡时间有明显的相关性,是推断早期人体死亡时间的有效的定量指标.     

应用单细胞凝胶电泳检测大鼠死后脾细胞核DNA降解与死亡时间的关系

目的:探讨应用单细胞凝胶电泳技术(SCGE)检测大鼠死后脾细胞核DNA降解与死亡时间的一般规律,为早期死亡时间的推断提供新的方法。方法:建立大鼠死亡模型,在死后27h内,每隔3h取脾组织样本进行单细胞凝胶电泳,用共聚焦显微镜摄取彗星图像,应用彗星图像分析软件(IMI1.0)进行图像分析,并作统计学分析。结果:大鼠死后,脾细胞在电泳图像上出现了明显的彗星形拖尾,其尾长(TL)、尾矩(TM)在一定的时间范围内(0～15h)随死亡时间的延长而逐渐增大,二者均与死亡时间(PMI)呈现一定的相关回归关系。结果:单细胞凝胶电泳技术可应用于早期死亡时间的推断;彗星图像分析软件为SCGE的结果分析提供了便利。     

青壮年猝死综合征脑干中缝背核5-羟色胺及5-羟色胺转运体免疫组化研究

目的 探讨5-羟色胺和5-羟色胺转运体在青壮年猝死综合征脑组织的表达及其意义.方法 运用免疫组织化学S-P法检测10例正常脑组织和8例青壮年猝死综合征脑组织中5-羟色胺和5-羟色胺转运体的表达水平,利用图像分析技术测量阳性细胞的平均光密度值.结果 青壮年猝死征死者脑干中缝背核5-羟色胺的平均光密度值低于正常脑组织,而5-羟色胺转运体的平均光密度值高于正常脑组织,差异均有统计学意义（P＜0.05）.结论 5-羟色胺及5-羟色胺转运体的变化参与青壮年猝死综合征的发病过程,可作为诊断青壮年猝死综合征的参考指标.     

兔急性肺损伤死后肝内血管内皮生长因子时序性变化的研究

目的研究大潮气量机械通气致兔急性肺损伤（ALI）12h后处死，死后肝微血管内血管内皮生长因子（VEGF）的动态变化。方法机械通气（VT60mL／kg，R50次／min）致急性肺损伤后12h处死兔，应用免疫组织化学染色（SABC法）和图像分析系统，观测死后不同时间点肝内微血管内皮细胞内vEGF的动态变化。结果对照组死后即刻肝内微血管内皮细胞胞浆vEGF呈弱阳性染色，死后2h阳性染色程度有轻度增加，但以后随PMI的延长VEGF逐渐减弱，死后18h呈阴性；试验组死后肝内微血管的内皮细胞胞浆VEGF呈强阳性表达，但随着PMI延长逐渐减弱，在死亡后0-24h减弱幅度较小，但21-36h阳性染色程度下降较快，直至死后42h随肝内微血管结构崩解而完全消失。结论大潮气量机械通气致急性肺损伤12h处死后，肝微血管内VEGF的变化在一定的时间段内与死亡时间呈现相关关系。     

P53基因在肝母细胞瘤中的表达及意义

目的 :探讨P5 3在肝母细胞瘤的表达情况。方法 :应用S -P法对 2 6例肝母细胞瘤行P5 3免疫组化染色 ,用显微图像分析仪测定阳性细胞百分率P/A(% )、平均光密度 (AOD)、积分光密度 (IOD)。结果 :P5 3在肝母细胞瘤中表达阳性率为 6 9.2 % (P <0 .0 1) ,P5 3的阳性表达水平 ,临床分期越晚越高 (P <0 .0 5 )。结论 :肝母细胞瘤的发生与P5 3蛋白异常表达有关 ,测定P5 3表达有助于判断预后。     

褪黑素对大鼠脑内强啡肽水平的影响

目的观察褪黑素（MLT）对大鼠脑内某些镇痛相关核团内神经细胞的强啡肽样免疫反应强度的影响。方法实验大鼠分给药组及对照组,分别腹腔注射MLT110mg/kg或配药液,1h后灌注取脑、冰冻切片,进行免疫组化染色,计算机图像处理技术测定染色脑片积分光密度（IOD）值。结果给药组大鼠视上核、中缝背核内强啡肽样免疫阳性反应减弱,其IOD值显著减少（P<0.01）;给药组大鼠下丘脑室旁核、中脑导水管周围灰质腹外侧区、中缝大核的IOD值,与对照组比较差别无显著意义。结论MLT可致视上核、中缝背核内强啡肽含量减少。     

兔死后角膜瞳孔区域图像颜色的时序性变化规律

目的探讨个体死后角膜瞳孔区域图像颜色时序性变化规律,为死亡时间（PMI）推断提供一种无损、客观方法。方法构建 家兔空气栓塞和溺死死亡模型,在温度20 ℃、湿度30%的暗室内,于死后72 h内每隔2 h用数码相机获取兔角膜图像。通过计 算机图像处理技术（MATLAB）分割出角膜瞳孔区域图像,提取RGBHSV 6项图像颜色特征参数值并进行与PMI关系的回归分 析,同时比较不同死因对死后角膜瞳孔区域图像颜色变化的影响。结果不同死因死后72 h内,兔角膜瞳孔区域图像的R、G、B 值均随PMI呈上升趋势,与PMI拟合关系较好（P<0.01）,H、S、V值与PMI关系均无统计学意义（P>0.05);经比较,两种死因死后 兔角膜瞳孔区域图像R、G、B值的变化趋势一致,与PMI的相关性相近,空气栓塞组兔角膜瞳孔区域图像的R、G、B实测值总体 大于溺死组。结论个体死后角膜瞳孔区域图像颜色变化随PMI呈一定规律性,死因会影响死后角膜瞳孔区域图像颜色特征参 数值大小。研究建立的相关回归方程为无损、客观推断PMI提供参考依据。     

MMP-9表达在不同方法获得破骨细胞的比较研究

目的比较基质金属蛋白酶9（matrix metailoproteinase 9,MMP-9）表达在机械法获得破骨细胞（osteoclasts,OC）和诱导法获得破骨样细胞（osteoclast like cells,OLC）的差异,为体外培养OC提供实验依据．方法采用机械分离方法,从1d龄sD大鼠四肢长骨获得成熟OC;利用RANKL（100ng／mL）和M—CSF（100ng／mL）诱导RAW264．7细胞形成OLC．采用免疫细胞化学和原位杂交技术检测不同方法获得的OC和OLC MMP-9蛋白及其mRNA的表达．结果诱导法12d组的积分光密度和平均光密度均明显高于机械法3d组．结论诱导12d的OLC MMP-9及其mRNA表达的明显高于机械法3d,为OLC提供了进一步的实验数据．