Effects of acute cold exposure on blood-brain barrier permeability in acute and chronic hyperglycemic rats

These experiments were carried out to study, the effects of cold exposure on the permeability of blood-brain barrier (BBB) in hyperglycemic rats. The integrity of the BBB was investigated using Evans blue albumin (EBA) extravasation. Serum glucose levels in hyperglycemic rats were significantly higher than that obtained from normoglycemic rats (P < 0.05). Mean arterial blood pressure in hypothermic groups significantly dropped into lower levels, than that obtained in normothermic groups (P < 0.05). The EBA extravasation to the cerebellum in the group of cold exposure+acute hyperglycemia significantly increased compared with the values obtained from the cold exposure group (P < 0.05). The EBA extravasation to the brain regions of diabetic rats exposed to cold increased more than that in normotermic control rats (P < 0.05), but did not exceed the levels in cold controls. The result of this study suggests that, acute hyperglycemia superimposed upon the permeability of BBB in the rat exposed to cold, only in selected regions of the brain, especially the cerebellum, and this result could be an important factor to explain the mechanisms of death related with hyperglycemia+cold exposure in forensic medicine.     

急慢性酗酒后脑组织Ngb、Hif-1α表达和Na^＋,K^＋-ATPase活性变化与TSAH的关系

目的探讨急慢性酗酒后脑组织脑红蛋白（Ngb）、缺氧诱导因子1α（Hif-1α）表达和Na＋,K＋-ATPase活性变化及与外伤性蛛网膜下腔出血（TSAH）的关系。方法雄性SD大鼠146只,随机分为急、慢性模型两大组,每组再各分为灌酒打击、灌水打击及单纯灌酒3个组,其中各打击组于最后一次灌胃后2h,用单摆式打击装置制作脑震荡模型;断颈处死大鼠,用免疫组化法观察脑组织Ngb、Hif-1α的表达、用分光光度法检测Na＋,K＋-ATPase。结果急、慢性灌酒打击组TSAH发生率分别为28.6%和82.4%;各灌酒打击和单纯灌酒组Ngb、Hif-1α-IOD值均显著高于各灌水打击组（P〈0.01）;急性单纯灌酒组2-4h Na＋,K＋-ATPas活性明显低于灌水打击组（P〈0.01）;慢性单纯灌酒组2h Na＋,K＋-ATPase活性高于急性灌酒组（P〈0.01）,而低于灌水打击组（P〈0.05）。结论急慢性灌酒后,脑组织Ngb、Hif-1α均呈反应性增强,Na＋,K＋-ATPase活性降低,酒精作用可能参与了TSAH的发生和死亡过程。     

MMP-2和MMP-9与脑损伤及其法医学意义

基质金属蛋白酶2和9（MMP-2、MMP-9）是基质金属蛋白酶（matrix metalloproteinases，MMPs）家族中的两个成员，可降解Ⅳ型胶原、层粘连蛋白等基质成分，从而使血脑屏障结构破坏，通透性增加，导致血管源性脑水肿，在脑血管疾病和脑损伤形成过程中起着重要的作用。在损伤后不同时段，MMP-2和MMPO含量及分布均呈现一定的波动性，探讨这些问题对脑损伤的治疗及在法医学中推断脑损伤时间有十分重要的意义。本文就这些问题的相关研究作一概述。     

甲基苯丙胺和乙醇在染毒大鼠体内联用的实验研究

目的研究甲基苯丙胺（MA）和乙醇联合应用在急性、亚急性染毒大鼠体内的情况。方法以MA1.0 mg/kg剂量对慢性自由饮酒大鼠多次腹腔注射,分别建立急性、亚急性染毒大鼠模型;处死后即时检测血中乙醇浓度、体液和组织样品中MA的浓度。结果多次注入MA药后,亚急性中毒大鼠体内的MA浓度高于急性中毒大鼠;急性和亚急性联合中毒大鼠体内MA浓度均高于急性和亚急性单一药物组。结论无论是否联用乙醇,14d内增加注射次数会在大鼠体内产生不同程度的MA残留;与乙醇联用可加速MA在大鼠体内的吸收,其药物浓度的升高程度随生物样品不同存在差异。     

The effects of sarin-like and soman-like organophosphorus agents on MAPK and JNK in rat brains

One sarin-like and one soman-like organophosphorus agent [bis(isopropyl methyl)phosphonate, BIMP and bis(pinacolyl methyl)phosphonate, BPMP] were injected intravenously (iv) in rats. An increase in the tyrosine phosphorylation of several proteins in the cytosol fraction of the brain was observed. Activation of c-Jun N-terminal kinase (JNK) and slight activation of mitogen-activated protein kinase (MAPK) in the cytosol were also observed. The activation of these enzymes may be related to the high toxicity of these nerve agents.     

Morphological signs of ethanol poisoning, alcohol abstinence and chronic alcoholic intoxication in the mesocorticolimbic dopaminergic system

Forensic medical diagnostics of ethanol poisoning, alcohol abstinence, and chronic alcoholic intoxication of the mesocorticolimbic dopaminergic system remains an unresolved problem and encounters difficulties. This situation is due not only to the marked vulnerability of the neurons of the dopaminergic system but also to the fact that its mechanisms are poorly understood. The objective of the present work was to substantiate and develop diagnostic criteria for ethanol poisoning, alcohol abstinence, and chronic alcoholic intoxication of the neurons both in the mesocorticolimbic dopaminergic system and in other brain regions. The object of the study was the brain of healthy adult subjects who died from alcohol intoxication (in the period of ethanol resorption) and under conditions of alcohol abstinence (completion of the abstinence course). The purpose of the study was to elucidate factors responsible for the different degree of damage to the neurons of various identification groups (intact, hypochromic, picnomorphic, shadow) and macrogliocytes. The cells of all these types were counted at an area of 0.25 sq. mm within 4 squares each having a side of 250 mcm in length. The absolute and relative number of neurons in each group as well as the number of polyneuronal satellite cells per one intact neuron was determined. It was shown that alcohol intoxication is associated with acute swelling of and severe damage to brain neurons caused by the combination of such factors as toxic effect of ethanol, excessive production of catecholamines, and functional overstrain of dopaminergic neurons. The severity of acute alcohol damage to the neurons decreased with the distance from the mid-brain dopaminergic nuclei. Restoration of neurons during alcohol abstinence was due to compensatory activation of interactions between neurons and glial cells. It decreased in the sequence from the paranigral nucleus of the ventral portion of mesencephalic tegumentum to the medial portion of the accumbence nucleus (field 24b, layer III of field la, layer V of field 1) depending on the initial severity of acute damage in the brain region being examined. The severity of damage to the neurons of the mesocorticolimbic dopaminergic system under conditions of chronic alcohol intoxication estimated from the number of shadow neurons was similar to the degree of acute swelling associated with ethanol poisoning and decreases from a maximum in the nuclei of the mesocorticolimbic dopaminergic system to a minimum in layer III of field 1.     

急性吗啡中毒大鼠主要器官内吗啡的免疫组化定位研究

一次静脉注射１２．５ｍｇ／ｋｇ。ｂｗ的盐酸吗啡染毒雄性Ｓ－Ｄ大鼠，２小时后处死，取其脑、肾、肝、肺、心组织以２％戊二醛和４％多聚甲醛混合液固定后，常规石蜡切片。运用抗吗啡抗血清及ＳＡＢＣ技术染色。结果显示上述组织切片有不同程度的阳性染色，阳性着色主要见于肾髓质部分肾小管上皮细胞，肝脏中央静脉周围的肝细胞、肺泡上皮细胞及肺内小支气管粘膜上皮细胞、中脑部分神经细胞、室管膜细胞、心肌细胞．以及各器官小血管及毛细血管内皮细胞胞浆、血浆及肾小管腔内尿液。     

The influence of ethanol on the level of ketone bodies in hypothermia

Presently available possibilities of macro- and microscopic diagnosis of death from hypothermia are very limited as the changes observed are either weakly specific (ecchymoses in the mucous membrane of the stomach, histological features of haemorrhagic pancreatic necrosis, cardiomyocyte necrosis or decreased content of glycogen in hepatocytes) or represent only local action of low temperatures (frostbites, violet patches in the region of knees and elbows, red livores) and they may not be present in cases of death from cooling at environmental temperature close to zero or higher.The study evaluated the usefulness of acetoacetic acid (Ac-Ac), beta-hydroxybutyric acid (beta-HBA) and acetone determinations in blood, urine and vitreous humour for diagnosis of death from hypothermia. These three substances called ketone bodies, are easily assimilated energetic substrates that get oxidized preferentially before glucose and fatty acids. In hypoglycaemia (also hypothermia-induced one), the tissues dependent on glucose (e.g. the brain) cover most of their energetic needs from oxidation of these compounds.The analysis of 16 cases of death in circumstances suggesting hypothermia (mainly of the alcohol abusers) showed that the degree of ketosis was inversely proportional to the blood ethanol concentration. This relation may result from stimulation of insulin release and a decrease in the release of its antagonists by ethanol, as well as from inhibition of free fatty acid (FFA) beta-oxidation due to increase in the NADH/NAD ratio. So, the antiketonaemic effects of ethanol (together with its influence on the dilatation of the peripheral vessels and inhibition of shivering thermogenesis by muscle relaxation), explain increased sensitivity of intoxicated persons to low temperatures.     

Comparison of blood-ethanol concentration in deaths attributed to acute alcohol poisoning and chronic alcoholism

Ethanol concentrations were measured in femoral venous blood in deaths attributed to acute alcohol poisoning (N = 693) or chronic alcoholism (N = 825), according to the forensic pathology report. Among acute alcohol poisonings were 529 men (76%) with mean age 53 years and 164 women (24%) with mean age 53 years. In the chronic alcoholism deaths were 705 men (85%) with mean age 55 years and 120 women (15%) with mean age 57 years. The blood-ethanol concentrations were not related to the person's age (r = -0.17 in acute poisonings and r = -0.09 in chronic alcoholism). The distribution of blood-ethanol concentrations in acute poisoning cases agreed with a normal or Gaussian curve with mean, median, standard deviation, coefficient of variation, and spread of 0.36 g/100 mL, 0.36 g/100 mL, 0.086 g/100 mL, 24% and 0.074 to 0.68 g/100 mL, respectively. The corresponding concentrations of ethanol in chronic alcoholism deaths were not normally distributed and showed a mode between 0.01 and 0.05 g/100 mL and mean, median, and spread of 0.172 g/100 mL, 0.150 g/100 mL, and 0.01 to 0.56 g/100 mL, respectively. The 5th and 95th percentiles for blood-ethanol concentration in acute poisoning deaths were 0.22 and 0.50 g/100 mL, respectively. However, these values are probably conservative estimates of the highest blood-ethanol concentrations before death owing to metabolism of ethanol until the time of death. In 98 chronic alcoholism deaths (12%) there was an elevated concentration of acetone in the blood (>0.01 g/100 mL), and 50 of these (6%) also had elevated isopropanol (>0.01 g/100 mL). This compares with 28 cases (4%) with elevated blood-acetone in the acute poisoning deaths and 22 (3%) with elevated blood-isopropanol. We offer various explanations for the differences in blood-ethanol and blood-acetone in acute poisoning and alcoholism deaths such as chronic tolerance, alcohol-related organ and tissue damage (cirrhosis, pancreatitis), positional asphyxia or suffocation by inhalation of vomit, exposure to cold coupled with alcohol-induced hypothermia, as well as various metabolic disturbances such as hypoglycemia and ketoacidosis.     

Blood and liver monoamine oxidases in fatal alcoholic intoxication]

Biochemical studies of monoamine oxygenases (MAO) were studied in albino rats and in subjects dead from ethanol poisoning, alcoholic cardiomyopathy, and coronary heart disease. Experiments demonstrated that chronic alcoholization leads to increase in the levels of both types of MAO (A and B), the longer the alcoholization, the more pronounced the increase. Intake of high ethanol doses in the course of regular alcohol abuse involves a drop in MAO activity (by 1.9-2-2 times). Study of MAO helps detect chronic alcoholization and differentiate it from coronary heart disease and alcoholic cardiomyopathy and thus make an objective conclusion about the cause of death and thanatogenesis.     

MOR1 receptor mRNA expression in human brains of drug-related fatalities-a real-time PCR quantification

The expression of the human micro-opiate receptor (MOR1) in post mortem human brain tissue was examined using real-time PCR technology. Tissue samples from 11 fatalities due to opiate overdose and five normal subjects with different causes of death were analysed in order to elucidate whether chronic opiate abuse is followed by a regulation of MOR1 expression. In each case nine selected brain regions (thalamus, caudate nucleus, hypothalamus, ventral tegmentum, hippocampus, amygdala, frontal cortex, nucleus accumbens, putamen) were evaluated. The MOR1-mRNA level was determined relative to the housekeeping gene beta2-microglobulin. While in most regions the MOR mRNA levels in the brain of addicts were not different from the control group-with varying levels between 0 and 15% of housekeeping gene level-in the brains of three drug-related fatalities an enormous increase was encountered in the thalamus where the MOR-mRNA level amounted for up to 10,000% of the measured housekeeping gene level. The results obtained by toxicological hair analysis in the group of drug-related fatalities indicate that the enormous thalamic MOR1-expression is primarily found in individuals who died from acute heroin overdose but did not show signs of a substantial chronic administration of the drug. Further studies have to be performed to evaluate if the observed MOR1-mRNA up-regulation in the thalamus in a subpopulation of acute lethal intoxications mirrors a state of functional hypersensitivity associated with the occurrence of death.     

大鼠急性脑干损伤神经细胞凋亡和轴突的变化

目的研究急性脑干损伤早期的病理变化,探讨其在急性脑干损伤中的法医学意义。方法采用自由落体造成大鼠急性脑干损伤模型,HE染色常规观察各部位脑组织的显微形态改变之后,用TUNEL末端标记检测神经细胞凋亡,用LSAB染色显示神经丝蛋白(NF)。结果实验动物模型能够较好的模拟法医案例;脑干损伤的大鼠脑组织淤血、水肿、环状出血,脑皮质内神经凋亡细胞数目明显增多(P<0.01);脑干部位神经轴突排列紊乱、肿胀、断裂。结论上述多种病理变化对急性脑干损伤的死后诊断具有一定的意义。     

甲基苯丙胺对血脑屏障毒性作用研究进展

甲基苯丙胺是一种苯丙胺类中枢神经系统兴奋剂,目前常出现此类药物滥用。血脑屏障是维持脑部微环境稳态,防止大脑受到致病性微生物侵袭及毒性分子作用的生理性动态屏障,其在维持大脑功能中作用非常重要。研究表明甲基苯丙胺能够通过血脑屏障对脑造成损害,但此过程中甲基苯丙胺是否损害血脑屏障及其机制尚不明确。本文对此进行综述,以期为相关研究提供依据参考。     

Creatine phosphokinase in serum and cerebrospinal fluid, and microscopic findings in brain and heart in hypothermic rabbits

Signs of hypothermia injury were studied in rabbits cooled to a core temperature of 30 degrees C by immersion in ice water and thereafter rewarmed to 35 degrees C. Anaesthetized control rabbits were kept normothermic (37 degrees C) for a corresponding time (4 h). Creatine phosphokinase (CPK) activity increased 24 h after hypothermia to 20-fold in serum. In cerebrospinal fluid the activity was already significantly (5-fold) increased after hypothermia and was still as high at 24 h. Smaller increase was also found in the control normothermic rabbits both in serum (10-fold) and cerebrospinal fluid (2-fold). The values had returned to the initial level after 1 week. Small haemorrhages were observed in the brain at 24 h and slight scarring was seen in the myocardium of some rabbits which had lived 4 weeks following hypothermia. The results indicate that CPK can be a useful marker in the diagnostics of hypothermia death, especially in cerebrospinal fluid, which is less affected than blood by autolysis.     

The role of ethanol abuse in the etiology of heroin-related death

Toxicology analyses and other forensic science data were used to examine the mechanisms through which ethanol increased the risk for death caused by injected street preparations of heroin. The authors studied 505 victims of fatal heroin overdose and compared subjects who had concentrations of blood ethanol greater than 1000 mg/L (n = 306) with those who had concentrations less than, or equal to 1000 mg/L (n = 199). We found significant negative correlations between concentrations of ethanol and morphine (a heroin metabolite) in blood (R2 = 0.11, P = 0.0001 for log10-transformed variables) as well as between concentrations of blood ethanol and bile morphine (R2 = 0.16, P = 0.0001 for log10 bile morphine versus blood morphine). Toxicologic evidence of infrequent heroin use was more common in decedents with blood ethanol concentrations greater than 1000 mg/L than in those with lower concentrations. Our data suggest that ethanol enhances the acute toxicity of heroin, and that ethanol use indirectly influences fatal overdose through its association with infrequent (nonaddictive) heroin use and thus with reduced tolerance to the acute toxic effects of heroin.     

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.     

Forensic medical significance of enzymatic products of ethanol oxidation in cadaveric brain

The concentrations of ethanol, acetaldehyde, and the oxidizing enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AIDH) were measured in neuronal cytoplasm, limbic cortical capillaries, and cardiovascular center of the medulla oblongata. The measurements were carried out by histochemical methods, gas-liquid chromatography, etc. The results were processed with consideration for the degree and stage of ethanol intoxication in case of death from ethanol poisoning and asphyxia in hanging. Increase of ethanol concentration in the blood was associated with a decrease and then increase in the brain concentrations of ADH and with an increase of AIDH concentration. Enzymatic changes predominated in capillary walls; the minimum shifts were observed in the neuronal cytoplasm of the cerebral limbic cortex, which confirms the neurohumoral nature of detoxication regulation. Lethal ethanol poisoning could occur during any stage of ethanol intoxication. The detected changes in ethanol, acetaldehyde, and metabolizing oxidoreductases in brain tissue can be used for forensic medical diagnosis of ethanol poisoning.     

Evaluation of human brain damage in fatalities due to extreme environmental temperature by quantification of basic fibroblast growth factor (bFGF), glial fibrillary acidic protein (GFAP), S100β and single-stranded DNA (ssDNA) immunoreactivities

Fatalities due to extreme environmental temperatures involving hypothermia (cold exposure) and hyperthermia (heat stroke) might present with poor or nonspecific morphological pathologies, which are insufficient to establish the cause of death in forensic practice. The present study immunohistochemically investigated basic fibroblast growth factor (bFGF), glial fibrillary acidic protein (GFAP), S100β and single-stranded DNA (ssDNA) in the parietal lobe and hippocampus of the brain in fatalities from hypothermia (n=15) and hyperthermia (n=18), and compared them to those of controls (n=39), including acute death due to ischemic heart disease, mechanical asphyxiation and drowning. In addition, S100β concentration in cerebrospinal fluid (CSF) was measured. Characteristic findings in hypothermia cases were higher glial bFGF immunopositivity in the cerebral cortex and white matter, and higher S100β immunopositivity in the cerebral cortex with a lower CSF S100β concentration. Hyperthermia showed lower glial GFAP and S100β immunopositivities in the white matter, and higher neuronal ssDNA immunopositivity in the cerebral cortex and hippocampus, accompanied by high glial bFGF and S100β immunopositivities in the cerebral cortex. These findings suggest neuroprotective glial responses without marked neuronal or glial damage in fatal hypothermia, and diffuse neuronal apoptosis despite initiation of neuroprotective cortical astrocyte responses, accompanied by glial damage in the white matter, in fatal hyperthermia. These markers may be useful for evaluating brain damage and responses in fatalities due to extreme environmental temperatures.     

Forensic medical diagnostics of acute and chronic alcohol intoxication

The principal characteristics and results of experimental studies on the problem of acute and chronic alcohol intoxication are presented. The mechanisms of toxic action of ethanol and acetaldehyde are considered with special reference to the comprehensive qualitative estimation of these toxicants and their pathomorphological effects in the target organs. The influence of ethanol-oxidizing enzyme systems in the brain on the development of alcohol tolerance is illustrated. The mechanisms of hormonal regulation via the hypothalamic-pituitary-adrenal axis in the case of exogenous alcoholemia are considered. The dependence of pathological morphological changes in the brain, hypophysis, and adrenal glands on the stage and severity of alcoholic intoxication has been elucidated. Criteria for the morphological and histochemical evaluation of the degree of alcohol-induced lesions and the severity of abstinence syndrome have been developed. The role of alcohol effects in tanatogenesis associated with alcohol-induced diseases is discussed.     

Multiple Symmetric Lipomatosis

A significant number of medicolegal deaths involve ethanol. Deaths may be related to the acute, intoxicating effects of ethanol, either in decedents or within persons responsible for causing the deaths of others. Additionally, deaths may be related to chronic alcoholism. A chronic alcoholic may display characteristic external features which allow an observer, such as a forensic pathologist or other physician, to conclude that he/she is probably an alcoholic. Herein, the authors report two decedents with a rare condition known as “multiple symmetric lipomatosis” (MSL), which has a strong correlation with chronic alcoholism. Identification of the peculiar features associated with MSL should prompt the forensic pathologist to consider chronic alcoholism as a probable diagnosis.