RyR2相关心脏性猝死的研究进展

雷诺定受体2(ryanodine receptor 2,RyR2)是心肌细胞肌浆网上主要的钙释放通道,主要参与心肌的兴奋收缩耦联过程,其基因突变及调控异常可导致心肌舒张期钙漏引起心律失常,甚至猝死。本文重点综述RyR2基因突变及调控异常引起心脏性猝死的机制,并对以后的研究提出建议,旨在为心脏性猝死的法医学诊断提供一种新的思路。     

Pathologic findings in malignant hyperthermia: a case report and review of literature

Malignant hyperthermia (MH) is a rare, potentially lethal disorder of skeletal muscle calcium homeostasis characterized by muscle contracture and life-threatening hypermetabolic crisis following exposure to halogenated anesthetics and depolarizing muscle relaxants. Susceptibility to MH results from mutations in calcium channel proteins that mediate excitation-contraction coupling, with the ryanodine receptor calcium release channel (RyR1) representing the major locus. The mode of inheritance appears to be autosomal dominant with variable penetrance. The authors report the death of a 60-year-old white male with a history of low back pain. He had undergone 2 back surgeries previously, the first occurring 10 years prior to his current presentation. Both previous procedures were done under generalized anesthetic with no complications. Recently, he developed stenosis and presented for fusion of vertebrae L3 and L4. The procedure was performed under general anesthetic including sevoflurane, with no intraoperative complications. The anesthesiologist noted that, near the end of the 2-hour procedure, the decedent's CO2 levels were slightly elevated. After the procedure, the decedent was extubated, the temperature probe which had been recording normal values was removed, and he was rolled from ventral to dorsal position. He immediately became hypotensive and bradycardic. Lifesaving interventions were begun. Subsequently, he went into cardiac arrest, at which time the temperature probe was reinserted into the trachea, where it read a body temperature of 109 degrees F. Malignant hyperthermia protocol was initiated, and interventions continued for over 2 hours, at which time they failed. At autopsy, the abdomen contained 1800 mL of blood, and bilateral hematomas were present in the psoas muscles. The authors present this case of clinically apparent malignant hyperthermia, discuss how to approach such a case, the gross and microscopic findings, ancillary studies, and a review of the literature.     

Post-mortem investigation of calcium content in liver, heart, and skeletal muscle in accidental hypothermia cases

The identification of hypothermia as cause of the death was always quite problematic in the field of forensic medicine. The aim of the present study was to verify the determination of calcium content in post-mortem liver, heart, and skeletal muscle samples as the biochemical marker defining hypothermia as the cause of death. The study involved 43 autopsy cases in which the circumstances of death indicated the effects of overcooling. The control group consisted of material collected from the corpses of 30 persons who were not exposed to low temperatures but died due to technical injuries (n = 5), asphyxia (n = 6), intoxication with ethanol and other substances (n = 8), and acute myocardial infarction/ischemia (n = 11). The concentration of calcium in autopsy samples was determined applying flame atomic absorption spectroscopy. Our study showed no significant differences of calcium content in tissues of persons who died due to hypothermia, over those who died in normothermic conditions.     

Blood factors of Sus scrofa following a series of three TASER electronic control device exposures

In a previous study, 18 repeated exposures of anaesthetized swine to an electro-muscular incapacitating device (TASER International's ADVANCED TASER X26 electronic control device) resulted in acidosis and increases in blood electrolytes. In the current study, experiments were performed to investigate effects of a more typical scenario of repeated exposures of the device on muscle contraction and changes in blood factors. Ten swine were exposed for 5s, followed by a 5-s period of no exposure, three times. Selected blood factors were monitored for 3h following exposure. Transient increases in blood glucose, lactate, sodium, potassium, calcium, and pCO(2) were consistent with previous reports in the literature dealing with studies of muscle stimulation or exercise. Blood pH was decreased immediately following exposure, but subsequently returned toward a normal level. Oxygen saturation (measured by pulse oximetry) was not changed significantly. In conclusion, three repeated TASER device exposures had only transient effects on blood factors, which all returned to pre-exposure levels, with the exception of hematocrit (which remained elevated after 3h). Since the increase in this factor was less than that which may occur after short periods of exercise, it is unlikely that this would be an indicator of any serious harm.     

Medicolegal studies on alcohol detected in dead bodies — alcohol levels in skeletal muscle

An experiment was carried out on rats to determine whether or not a skeletal muscle sample was suitable for the determination of ethanol concentration in a carcass. Gas chromatography was used to estimate the ethanol and n-propanol concentrations in the femoral muscle and intracardial blood. The ethanol concentration of each sample was corrected according to the moisture ratio of circulating blood, viz., 78.5%.The ethanol concentration ratio of blood to muscle was 1.03 two hours after ethanol administration. When the carcasses of rats pre-treated with ethanol were stored at 15 °C and 25 °C, respectively, the ethanol concentrations in muscle and blood increased with time. At all times the concentration was higher in blood than in muscle, and also higher in samples collected from the carcass stored at 25 °C than at 15 °C.When the control carcass was stored in the same manner, the postmortem production of ethanol was noticed in both blood and muscle. As in the experimental rats, the control rats exhibited a higher blood ethanol than muscle ethanol level. Again, the ethanol concentration was higher in samples collected from the carcass stored at 25 °C than at 15 °C. The ratio of ethanol to n-propanol was less than 20:1 in blood and less than 10.1 in muscle.These results suggest that skeletal muscle may be a suitable tissue for the postmortem detection of ethanol.     

Verapamil toxicity: an unusual case report and review of the literature

Verapamil blocks the rapid influx of calcium into the cardiac myocytes of the cardiac conduction system and smooth muscle of the vasculature, resulting in decreased myocardial contractility, prolonged conduction time, and vascular relaxation. A sustained-release form, verapamil SR (or ER), is available that contains higher levels of medication and requires only once-daily dosing. The majority of reported fatal cases of verapamil toxicity are due to massive, intentional overdoses. Herein, we present an unusual case of fatal verapamil SR toxicity in a 57-year-old female that resulted from accidental overdose of only 3 tablets (720 mg), as witnessed by the decedent's daughter. In spite of the low dose ingested, the postmortem cardiac blood verapamil level was clearly toxic (6000 ng/mL, or 6 mg/L). Her preexisting medical conditions included hypercholesterolemia, hypertension, iron deficiency anemia, diabetes mellitus, and associated mild chronic renal failure. Complicating factors, which likely include the decedent's preexisting renal and cardiac disease, and a review of the available literature will be discussed.     

Evaluation of postmortem serum calcium and magnesium levels in relation to the causes of death in forensic autopsy

There appears to be very poor investigation of postmortem serum calcium (Ca) and magnesium (Mg) for diagnostic evidence to determine the cause of death. The aim of the present study was a comprehensive analysis of the serum levels in relation to the causes of death in routine casework. Autopsy cases (total, n=360; 5-48 h postmortem), including blunt injury (n=76), sharp injury (n=29), asphyxiation (n=42), drownings (n=28: freshwater, n=11; saltwater, n=17), fire fatalities (n=79), methamphetamine (MA) poisoning (n=8), delayed death from traumas (n=37), and acute myocardial infarction/ischemia (AMI, n=61), were examined. In total cases, there was no significant postmortem time-dependent rise in serum Ca and Mg. Both Ca and Mg levels in the heart and peripheral blood were significantly higher in saltwater drowning compared with those of the other groups. In addition, a significant elevation in the Ca level was observed in freshwater drowning and fire fatalities, and in the Mg level in fatal MA intoxication and asphyxiation. Topographic analyses suggested a rise in serum Ca and Mg due to aspirated saltwater in drowning, that in serum Ca in freshwater drowning and fire fatalities of peripheral skeletal muscle origin and that in serum Mg in MA fatality and asphyxiation of myocardial and/or peripheral origin. These markers may be useful especially for diagnosis and differentiation of salt- and freshwater drownings and may be also helpful to determine the causes of death involving skeletal muscle damage, including burns and MA intoxication.     

Paraquat myopathy: report on two suicide cases.

We describe two suicide cases in which old paraquat was ingested. In conjunction with lung involvement a pronounced degeneration was observed in skeletal muscle of one who died on the 14th day after the ingestion. The following sarcoplasmic or endoplasmic reticulum Ca2+ ATPase (SERCA) monoclonal antibodies were used for skeletal muscle fiber typing by an immunohistochemical method: NCL-SERCA1, reactive with type 2 fiber (fast-twitch), and NCL-SERCA2, reactive with type 1 fiber (slow-twitch). The examination revealed that the remarkably degenerated fibers belonged to type 1 muscle fibers. This case showed an abrupt increase of plasma CK levels (1796 mU/ml) on the fifth day after the ingestion. The authors presume that the damage to the skeletal muscle had occurred in this period. The degeneration of the muscle seemed to be attributable to the long retention of paraquat in the tissue because these findings were not observed in the other case who died on the fifth day. Paraquat-induced myopathy may develop in prolonged paraquat poisoning. The examination of CK levels in plasma will be useful for diagnosis of damage of skeletal muscle.     

大鼠骨骼肌挫伤愈合过程中caspase-3的表达及时间规律

目的观察大鼠骨骼肌挫伤愈合过程中,caspase-3的表达及其变化规律。方法制作大鼠右小腿骨骼肌挫伤模型,应用免疫组织化学法、Western blot法及caspase-3活性测定法对不同时间段大鼠骨骼肌挫伤后caspase-3表达进行检测。结果免疫组织化学及Western blot均显示,挫伤周边区caspase-3的活性随时间延长逐渐升高,5d时达最高峰,随后活性逐渐下降。免疫组织化学染色结果显示,伤后3～5d挫伤处坏死的骨骼肌细胞被清除干净,新形成的肌细胞大量增生,且新形成的肌细胞胞浆内有caspase-3表达;损伤后7～14d,新形成的肌细胞内仍然有caspase-3的阳性表达。结论大鼠骨骼肌挫伤后,挫伤周边骨骼肌表达caspase-3有一定的时间规律性,可望作为推断骨骼肌挫伤时间的指标之一;大鼠骨骼肌挫伤后,新形成的肌细胞表达caspase-3,提示caspase-3可能在骨骼肌分化过程中起重要作用。     

多层螺旋CT冠状动脉钙化积分在评价冠心病猝死中的应用

目的通过运用多层螺旋CT（multi—slicespiralcomputedtomography,MSCT）冠状动脉钙化积分（coronaryarterycalciumscoring．CACS）的方法．评价其在冠心痛（coronaryarterydisease,CAD）猝死案例中的法医学应用价值,为虚拟解剖鉴定CAD猝死探索有效手段。方法收集9例进行法医学鉴定的心源性猝死案例．尸体解剖前均进行MSCT扫描。通过Agatston’s法对每例冠状动脉钙化程度进行定量分析,并计算CACS,钙化积分〉400作为存在CAD的评判标准,并与尸体解剖结果比较。结果经尸体解剖证实CAD猝死的9例案例中仅有2例钙化积分〉400,CACS对CAD的预测率仅为22．2％。MSCT检查与系统尸体解剖均发现CAD猝死案例中普遍存在不同程度肺水肿改变,冠状动脉左前降支较其他分支发生管腔狭窄的发病率高。结论运用MSCT并结合计算CACS可对CAD猝死案例中冠状动脉钙化明显的进行检测,而钙化不明显的需要辅以如尸体血管造影等其他技术。     

Vitreous humor biochemical constituents: evaluation of between-eye differences

The present study was conducted to investigate the differences in the vitreous humor biochemical concentrations for vitreous electrolytes and calcium in the same pair of eyes at identical postmortem interval (PMI). The vitreous humor samples were collected independently in both eyes from 48 autopsies (PMI range, 4.5-84.3 hours) with documented time of death. The samples were analyzed for potassium, sodium, chloride, and calcium using a Beckman Coulter LX20 Automated Analyzer based on ion-selective electrode methodology. There were no statistically significant between-eye differences at identical postmortem interval. A significantly high correlation was observed between paired potassium concentrations of both the eyes. A highly significant linear correlation was observed between the individual eye and mean potassium concentrations of both the eyes with postmortem interval. The observed differences were not significantly correlated with postmortem interval. The results demonstrated that the between-eye differences for vitreous electrolytes and calcium are insignificant. Therefore, the utility of vitreous biochemistry, particularly potassium in postmortem interval estimation and other forensic applications, cannot be questioned solely on the basis of these differences.     

Determination of calcium and magnesium in postmortem human vitreous humor as a test to ascertain the cause and time of death

The concentrations of magnesium and total calcium human vitreous humors obtained postmortem was tested. In the groups of heart disease and asphyxia a correlation between the postmortem interval and calcium or magnesium concentrations was found. There was no correlation between postmortem interval and calcium respectively magnesium in the other causes of death. It seems possible that the analysis of vitreous humor may be useful in both diagnosis and determination of postmortem interval in the cases of heart death and asphyxia.     

Experimental tests for the validation of active numerical human models

The development of numerical human models is a topic of current interdisciplinary research. In the field of automotive safety these models can be applied for the optimization of protection systems. In forensic research human models can be used for the investigation of injury mechanisms and for the prediction and reproduction of injury patterns. However, up to now human models have been validated on the basis of PMHS tests without considering the effects of muscle activity. This paper shows two experimental volunteer test set-ups for the generation of experimental validation data. In a pendulum set-up the influence of muscle activity on the human kinematics was investigated. A drop test set-up was developed for the analysis of the effects of muscle activity on impact response characteristics of muscle tissue. Experimental results, presented in this paper, can be used for the validation and optimization of active numerical human models.     

大鼠骨骼肌钝挫伤分级模型的建立

目的建立一个控制性、重复性好,并可分级的骨骼肌挫伤动物模型。方法采用自行设计的机械式撞击装置,以撞击速度（velocity,v）和形变（deformation,DF）作为外力参数,并保持较恒定的撞击时间,使击锤打击瞬间速度不同,造成轻、中、重不同程度的大鼠腓肠肌挫伤。结果在肉眼、HE染色观察下．39只大鼠腓肠肌出现可分级的病理学改变。在低撞击水平（v：2m／s,DF：5．5mm）,轻度挫伤组病理改变局限于大鼠腓肠肌肌外膜和腓肠肌表层部位;在中度撞击水平（v：2．5m／s,DF：6．5mm）,中度挫伤组病理改变可见于大鼠腓肠肌肌外膜和大部分腓肠肌;在高撞击水平（v：3m／s,DF：7,5mm）,重度挫伤组病理改变除中度挫伤组累及部位外,还伤及比目鱼肌。结论该模型制作出常见的钝力性骨骼肌挫伤,以撞击速度和形变作为外力参数,成功建立了机械骨骼肌挫伤分级模型,致伤程度较一致,是骨骼肌挫伤研究较合适的动物模型,为深入研究钝力性骨骼肌挫伤奠定了基础．     

中介素（IMD）对大鼠心肌细胞缺氧-复氧损伤的作用

目的探讨中介素（IMD）对大鼠体外培养心肌细胞缺氧-复氧损伤的作用。方法用大鼠H9c2心肌细胞株制作实验模型,样本分为对照组、缺氧-复氧组（缺氧1h、复氧30min）、IMD组（缺氧-复氧前30min加入10-7mol/L IMD）。采用MTT比色法检测心肌细胞活力;透射电镜观察细胞超微结构;激光共聚焦显微镜观察并测定细胞内钙离子浓度;流式细胞术测定细胞凋亡率。结果与对照组比较,缺氧-复氧组和IMD组细胞存活率显著降低,而IMD处理组明显升高细胞存活率（P〈0.01）;在形态学上,IMD预处理可明显减轻缺氧-复氧对大鼠心肌细胞的损伤;缺氧-复氧组细胞[Ca2＋]i荧光强度和细胞凋亡率比对照组显著升高,IMD预处理可明显降低上述升高的比率（P〈0.01）。结论 IMD对大鼠心肌细胞缺氧-复氧损伤有一定保护作用,提高心肌细胞存活率、减轻心肌细胞钙超载和抑制凋亡是其作用途径。     

Freezing skeletal muscle tissue does not affect its decomposition in soil: Evidence from temporal changes in tissue mass,microbial activity and soil chemistry based on excised samples

The study of decaying organisms and death assemblages is referred to as forensic taphonomy, or more simply the study of graves. This field is dominated by the fields of entomology, anthropology and archaeology. Forensic taphonomy also includes the study of the ecology and chemistry of the burial environment. Studies in forensic taphonomy often require the use of analogues for human cadavers or their component parts. These might include animal cadavers or skeletal muscle tissue. However, sufficient supplies of cadavers or analogues may require periodic freezing of test material prior to experimental inhumation in the soil. This study was carried out to ascertain the effect of freezing on skeletal muscle tissue prior to inhumation and decomposition in a soil environment under controlled laboratory conditions. Changes in soil chemistry were also measured. In order to test the impact of freezing, skeletal muscle tissue (Sus scrofa) was frozen (?20 °C) or refrigerated (4 °C). Portions of skeletal muscle tissue (～1.5 g) were interred in microcosms (72 mm diameter × 120 mm height) containing sieved (2 mm) soil (sand) adjusted to 50% water holding capacity. The experiment had three treatments: control with no skeletal muscle tissue, microcosms containing frozen skeletal muscle tissue and those containing refrigerated tissue. The microcosms were destructively harvested at sequential periods of 2, 4, 6, 8, 12, 16, 23, 30 and 37 days after interment of skeletal muscle tissue. These harvests were replicated 6 times for each treatment. Microbial activity (carbon dioxide respiration) was monitored throughout the experiment. At harvest the skeletal muscle tissue was removed and the detritosphere soil was sampled for chemical analysis. Freezing was found to have no significant impact on decomposition or soil chemistry compared to unfrozen samples in the current study using skeletal muscle tissue. However, the interment of skeletal muscle tissue had a significant impact on the microbial activity (carbon dioxide respiration) and chemistry of the surrounding soil including: pH, electroconductivity, ammonium, nitrate, phosphate and potassium. This is the first laboratory controlled study to measure changes in inorganic chemistry in soil associated with the decomposition of skeletal muscle tissue in combination with microbial activity.     

Development of rigor mortis is not affected by muscle volume

There is a hypothesis suggesting that rigor mortis progresses more rapidly in small muscles than in large muscles. We measured rigor mortis as tension determined isometrically in rat musculus erector spinae that had been cut into muscle bundles of various volumes. The muscle volume did not influence either the progress or the resolution of rigor mortis, which contradicts the hypothesis. Differences in pre-rigor load on the muscles influenced the onset and resolution of rigor mortis in a few pairs of samples, but did not influence the time taken for rigor mortis to reach its full extent after death. Moreover, the progress of rigor mortis in this muscle was biphasic; this may reflect the early rigor of red muscle fibres and the late rigor of white muscle fibres.     

Estimation of age at death based on quantitation of the 4977-bp deletion of human mitochondrial DNA in skeletal muscle

The 4977-bp deletion in human mitochondrial DNA (mtDNA) is known to accumulate in various tissues with age. Since this deletion in mtDNA correlates closest with age in muscle tissue, iliopsoas muscle tissue was taken at autopsy from 50 persons aged 24-97 years to determine whether age at death can be estimated based on the amount of the 4977-bp deletion in skeletal muscle. Total DNA (nuclear and mtDNA) was extracted from 100 mg tissue and the 4977-bp deletion quantified using a kinetic polymerase chain reaction (PCR) followed by visualization of the products on silver stained polyacrylamide gels. The amount of the 4977-bp deletion of mtDNA ranged from 0.00049% to 0.14% depending on age, with a correlation coefficient of r = 0.83 (P = 0.0001). In forensic practice this method can aid in the estimation of age at death with a relatively wide confidence interval, thus enabling a discrimination between young and elderly persons in the identification of human remains based solely on skeletal muscle.     

大鼠尺神经的损伤修复对其支配的爪内骨骼肌形态学改变的影响

目的通过对大鼠尺神经不同损伤断面和不同修复时间的实验研究．观察其支配的爪内骨骼肌形态学的改变,探讨法医临床学对尺神经损伤鉴定的最佳时机。方法选用SD大鼠42只．建立尺神经损伤修复模型,取爪内骨骼肌称量湿重,经HE和Masson染色,测量肌肉湿重、肌细胞横截面积和胶原纤维占肌纤维截面积的比率。结果（1）尺神经损伤后早期修复组的爪内骨骼肌萎缩程度均低于实验对照组．且即刻修复组比1个月修复组爪内骨骼肌功能恢复效果好。（2）失尺神经支配后爪内骨骼肌中的胶原纤维逐渐增多。（3）腕部与肘部尺神经损伤后,在相同时间修复尺神经,爪内骨骼肌萎缩的变化规律一致。结论尺神经损伤即刻修复能完全恢复爪内骨骼肌形态学改变,1个月修复能部分恢复其形态学改变．3个月修复则无法恢复其形态学改变;爪内骨骼肌中胶原纤维的增多是影响手术疗效和功能恢复的可能性原因之一;腕部与肘部的尺神经损伤修复对爪内骨骼肌形态学改变的影响无明显差异。     

大鼠死后心肌骨骼肌细胞肌动蛋白变化及与死亡时间的关系

目的　探讨大鼠死后心肌骨骼肌细胞肌动蛋白的变化及其与死亡时间的关系。方法　采用免疫组化S P法和IBSA图像分析系统 ,观测大鼠死后不同时间心肌骨骼肌抗肌动蛋白单克隆抗体 (HHF3 5 )染色变化。结果　在大鼠死后 5 4h内 ,心肌骨骼肌呈不同程度的HHF3 5缺染 ,其面积随死后时间的延长而增大。经对IBSA图像分析数据进行方差分析 (F心肌 =5 88 2 7,F骨骼肌 =3 5 1 2 5 ,P <0 0 5 ) ,具有显著性差异 ;经逐步回归分析 (r心肌 =0 943 ,r骨骼肌 =0 95 8,P <0 0 5 ) ,具有正相关关系。所建方程y心肌 =-3 0 5 68+1 0 0 3x1,y骨骼肌 =-2 4 897+0 986x2 (X为HHF3 5缺染面积均数 )具有统计学意义。结论　在一定时间内 ,大鼠死后不同时间心肌骨骼肌细胞HHF3 5缺染面积 ,随死后时间的延长而增大。