Tissue distribution of olanzapine in a postmortem case

Olanzapine is a relatively new antipsychotic drug used in the United States for the treatment of schizophrenia. Since its release in the United States market in 1996, few cases of fatal acute intoxication have been reported in the literature. This article describes the case of a 25-year-old man found dead at home who had been prescribed olanzapine for schizophrenia. This case is unique because of the measurement of olanzapine in brain tissue obtained from seven regions in addition to the commonly collected biologic matrices. Olanzapine was detected and quantitated by basic liquid-liquid extraction followed by dual-column gas chromatographic analysis with nitrogen phosphorus detection. The assay had a limit of detection of 0.05 mg/L and an upper limit of linearity of 2 mg/L. The presence of olanzapine was confirmed by gas chromatography-mass spectrometry by use of electron impact ionization. The concentrations of olanzapine measured in this case were as follows (mg/L or mg/kg): 0.40 (heart blood), 0.27 (carotid blood), 0.35 (urine), 0.61 (liver), negative (cerebrospinal fluid), 0.33 mg in 50 ml (gastric contents). In the brain, the following distribution of olanzapine was determined (mg/kg): negative (cerebellum), 0.22 (hippocampus), 0.86 (midbrain), 0.16 (amygdala), 0.39 (caudate/putamen), 0.17 (left frontal cortex), and 0.37 (right frontal cortex). The cause of death was determined to be acute intoxication by olanzapine, and the manner of death was accidental.     

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.     

毒鼠强中毒大鼠脑神经元GABA_A α1受体的检测

目的探讨GABAAα1受体在毒鼠强中毒大鼠脑组织中的变化及其机制。方法选择健康Sprague-Daw-ley大白鼠30只,分成5组,每组6只;分别以2.0 LD50、1.0 LD50、1/2 LD50、1/10 LD50毒鼠强量,采用灌胃染毒方法制作毒鼠强中毒模型,并以健康大鼠灌服生理盐水为对照;断颈处死大鼠,提取脑皮质、海马及脑干脑桥等脑组织进行免疫组织化学染色,观察GABAAα1受体阳性染色神经元的变化。结果不同LD50量染毒大鼠的皮层、海马及脑干,其GABAAα1受体免疫组化阳性染色神经元较对照组组减少,其中以1.0 LD50、1/2LD50组减少最为显著;相同染毒剂量,以脑干组织减少最多。结论GABAAα1受体表达下降与毒鼠强中毒机制相关。     

An attempt at objectivity of post mortem diagnostic of brain oedema

The investigations were carried out on 99 brains of which 83 exhibited macroscopic features of brain oedema. The tissue investigated came from four regions of the brain: the cortex of frontal and occipital regions, the white matter and the tissue of the pons varioli. The percentage of water in these regions was evaluated and the MIB test (methyl iodide in benzene) was used for a part of the analysed material (60 cases). Considerable differences in water percentage were found between the cortical layer and white matter and the tissue of the pons varioli.     

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.     

大鼠脑损伤分级自由落体打击模型的建立

目的建立一个控制性与重复性好,并可分级的脑损伤动物模型。方法根据自由落体原理,使击锤从不同的高度下落,造成轻、中、重不同程度的大鼠脑损伤,肉眼、光镜观察损伤的程度。结果肉眼、光镜观察下出现可分级的病理学改变。轻度损伤组病理改变局限于大鼠大脑皮质浅层,中度损伤组病理改变可见于大脑皮质及深部白质内的基底核、海马、胼胝体、丘脑,重度损伤组病理改变除中度损伤累及部位外,还累及脑干。挫伤灶周围神经元变性、坏死范围和程度随损伤程度的加重而增大。各组内每只动物所受损伤程度一致。结论该模型制作出常见的闭合性颅脑加速伤损伤,致伤程度较一致,重复性好,可分级,是脑损伤实验研究较合适的动物模型。     

大鼠脑液压冲击伤caspase-8的表达

目的探讨大鼠脑损伤后caspase-8表达情况,为脑损伤的诊断及损伤时间推断提供依据。方法建立大鼠脑液压冲击伤模型。用免疫组化与图像分析技术分别检测伤后15,30min和1,3,6,12h及1,4,7,14d大鼠皮质、丘脑、海马等部位caspase-8的表达。结果发现伤后30min大脑皮质和海马caspase-8开始出现表达,随时间增加其表达亦逐渐增加,伤后3h显著增加,24h达到高峰,4d后逐渐减少,14d基本恢复正常;而丘脑在伤后1h才开始出现阳性表达,伤后6h出现明显表达,24h达到高峰,4d后逐渐减少,14d基本恢复正常。同时发现损伤对侧海马及丘脑出现相应的变化规律。结论caspase-8阳性表达的变化规律可作为脑损伤诊断及损伤时间推测的指标之一。     

大鼠脑挫伤后脑组织COX-1/COX-2的表达

目的　观察脑损伤后COX 1和COX 2蛋白表达及其时序性变化 ,探讨脑损伤的分子机制及法医学脑损伤时间推断。方法　以自由落体撞击雄性SD大鼠右顶叶制备脑挫伤模型 ,用免疫组织化学SP法处理脑组织检材 ,观察不同时间 ( 1、 3、 5、 7、 14d)脑组织COX 1/COX 2蛋白的表达情况。结果　正常及手术对照组大鼠 ,脑组织内有低水平的COX 1/COX 2表达 ;脑挫伤后 1～ 5d ,大鼠脑组织内COX 1表达逐渐增加 ,14d时仍维持在高表达水平 ;脑挫伤后 1～ 3d ,大鼠脑组织皮质内COX 2表达逐渐增加 ,1d时海马COX 2表达达高峰。结论　脑挫伤后可诱导COX 1/COX 2蛋白在脑内表达 ,并呈现出时序性变化。     

Quantitative immunohistochemical analysis of human brain basic fibroblast growth factor, glial fibrillary acidic protein and single-stranded DNA expressions following traumatic brain injury

Previous studies demonstrated the healing process after traumatic brain injury (TBI), usually at the site or in the area adjacent to the injury, in connection with wound timing. However, the whole brain condition after TBI has not been elucidated clearly. In the present study, we investigated immunohistochemistry of the basic fibroblast growth factor (bFGF), glial fibrillary acidic protein (GFAP) and single-stranded DNA (ssDNA) in the parietal lobe and hippocampus distant from the primary injury site in forensic autopsy cases of TBI (n=174). Characteristic findings were detected with regard to brain compression signs and survival time (ST). Peracute deaths (n=22) had a lower GFAP positivity in the parietal white matter. Fatalities without a brain compression sign (parahippocampal herniation/Duret hemorrhage; n=53) had a lower brain weight without glial loss; however, GFAP positivity in the parietal white matter was decreased during ST of <12h, and glial bFGF positivity was increased at each site in deaths after 12h to 3 days, followed by a delayed neuronal loss after 3 days. Fatalities with a brain compression sign (n=78) showed a higher brain weight, and gradual glial and neuronal losses with an early increase of glial bFGF positivity in the parietal cerebral cortex (ST <0.5h). This was followed by an increase of glial bFGF positivity in other sites (ST, 6-24h), and final decreases of glial bFGF and GFAP positivities with increased neuronal ssDNA positivity in the parietal lobe and hippocampus (ST >3 days), which were detected in earlier deaths despite decompressive craniectomy (ST, 12-60h; n=21). These observations suggest that the combined use of bFGF, GFAP and ssDNA immunohistochemistry can be used to evaluate the severity of damage and response of brain after TBI.     

Distribution of Enantiomers of Methadone and Its Main Metabolite EDDP in Human Tissues and Blood of Postmortem Cases

Knowledge concerning the distribution of methadone in postmortem human tissue and the effect of postmortem redistribution on methadone is today limited making the choice of a suitable substitute for femoral blood difficult when this is not available. Cardiac blood, femoral blood, muscle, and brain tissue concentrations of the enantiomers of methadone and its metabolite 2‐ethyl‐1,5‐dimethyl‐3,3‐diphenylpyrrolinium were recorded for 155 postmortem cases. Brain and muscle tissue concentrations exceeded the femoral blood concentrations with a median fold of 2.3 and 1.6, respectively, but both had a better correlation than cardiac blood to femoral blood concentrations. The Kruskal–Wallis test showed a significant dependency on time and body mass index for some of the matrix ratios over femoral blood. We conclude brain or muscle tissue may constitute a better alternative for measurement of methadone than cardiac blood for situations in which femoral blood is not available, despite concentrations in both matrices being systematically higher.     

The expression of excitatory amino acid transporter 2 in traumatic brain injury

It is well recognized that glutamate is the major excitatory neurotransmitter, which is removed from the synaptic cleft by excitatory amino acid transporter 2 (EAAT2) located on the perisynaptic astrocytes and that neuronal death has been associated with an increased extracellular glutamate concentration. In this study, we have immunohistochemically demonstrated the expression of EAAT2 protein in the human brain after traumatic brain injury (TBI). The EAAT2 expression patterns can be divided into three types: continuous and highly extensive staining (E); continuous but sporadic staining (M); and sporadic pattern staining (S). In six of the nine short survival cases studied (1 h to 1 day), continuous and highly extensive staining for EAAT2 (E type) was observed in the ipsilateral cerebral cortex. On the other hand, we were able to demonstrate weak staining (S and M types) in 5 of the 7 long survival cases (≥1 day) and in 12 of the 14 very short survival cases (<1 h) studied. Similar findings were obtained in the contralateral cerebral cortex and also in the ipsilateral hippocampus. In addition, positive staining for glial fibrillary acidic protein was detected around the cerebral contusion, but the EAAT2-positive expression was not observed in the same region for all of the six short and long survival cases (≥1 h) after TBI. These findings clearly showed the differences in EAAT2 expression in the cerebral cortex according to the survival time and severity of cerebral contusion after TBI. Therefore, we emphasized that EAAT2 might play an important role in contributing to extracellular glutamate concentrations and secondary brain injury after TBI.     

The expression of excitatory amino acid transporter 2 in traumatic brain injury

It is well recognized that glutamate is the major excitatory neurotransmitter, which is removed from the synaptic cleft by excitatory amino acid transporter 2 (EAAT2) located on the perisynaptic astrocytes and that neuronal death has been associated with an increased extracellular glutamate concentration. In this study, we have immunohistochemically demonstrated the expression of EAAT2 protein in the human brain after traumatic brain injury (TBI). The EAAT2 expression patterns can be divided into three types: continuous and highly extensive staining (E); continuous but sporadic staining (M); and sporadic pattern staining (S). In six of the nine short survival cases studied (1 h to 1 day), continuous and highly extensive staining for EAAT2 (E type) was observed in the ipsilateral cerebral cortex. On the other hand, we were able to demonstrate weak staining (S and M types) in 5 of the 7 long survival cases (> or =1 day) and in 12 of the 14 very short survival cases (<1 h) studied. Similar findings were obtained in the contralateral cerebral cortex and also in the ipsilateral hippocampus. In addition, positive staining for glial fibrillary acidic protein was detected around the cerebral contusion, but the EAAT2-positive expression was not observed in the same region for all of the six short and long survival cases (> or =1 h) after TBI. These findings clearly showed the differences in EAAT2 expression in the cerebral cortex according to the survival time and severity of cerebral contusion after TBI. Therefore, we emphasized that EAAT2 might play an important role in contributing to extracellular glutamate concentrations and secondary brain injury after TBI.     

脑震荡大鼠脑组织MDA、SOD、TNF-a及IL-1β表达变化

目的观察脑震荡大鼠脑组织中丙二醛（malondialdehvde,MDA）含量、超氧化物歧化酶（superoxidedismutase,SOD）活性及肿瘤坏死因子-ol（tumornecrosisfactor-a,TNF-a、白细胞介素-1β（interleukin-1β,IL-1β表达的变化,探讨脑震荡后继发性脑损伤机制。方法建立大鼠脑震荡模型,Weil氏染色观察大鼠脑组织病理改变：光化学分析方法检测脑组织内MDA含量、SOD活性;免疫组化法检测大脑皮质和海马区TNF-a、IL-1β表达。结果Weil氏染色显示神经髓鞘排列紊乱,弯曲肿胀,12h后更加明显;脑震荡后大鼠脑组织MDA含量较对照组明显升高．而SOD活性较对照纽明显降低;脑震荡后大鼠皮质及海马区细胞胞浆中TNF-a、IL-1β较对照组表达量明显上调。结论脑震荡大鼠脑组织存在氧化应激和炎性损伤,可能在脑震荡后继发性脑损伤中起重要作用。     

A review of methadone deaths in Jefferson County,Alabama

Interpretation of the concentration of a drug is more difficult when a combination of drugs is present in a decedent's blood. An increase in deaths resulting from co-intoxication with methadone and a benzodiazepine led the authors to perform a retrospective study of cases examined at the Jefferson County Coroner/Medical Examiner Office. They found 101 deaths wherein methadone was detected in the blood. Based on the drugs detected in the blood, these 101 cases were grouped into four categories: (1) pure methadone intoxication, (2) intoxication with methadone and benzodiazepine, (3) intoxication with methadone and other drugs excluding benzodiazepine, and (4) intoxication with methadone, benzodiazepines, and other drugs. Methadone was the sole intoxicant in 15 cases, with a mean concentration of 0.27 mg/L. Benzodiazepines were the most frequently detected co-intoxicant (60 of 101 cases). Benzodiazepine was the only co-intoxicant in 30 cases, and the mean methadone concentration in those 30 cases was 0.599 mg/L. Higher levels of methadone may occur in acute intoxication with methadone and benzodiazepine because benzodiazepines compete with methadone for methadone receptors. Higher levels of methadone may occur with chronic abuse of methadone and benzodiazepines because over time, benzodiazepines inhibit the hepatic enzymes that metabolize methadone.     

Blood, brain, and hair GHB concentrations following fatal ingestion

Despite the increasing incidence of illicit use of gamma-hydroxybutyrate (GHB), little information is available documenting levels of the drug in GHB fatalities. We measured GHB levels in postmortem blood, brain and hair specimens from a suspected overdose case by gas chromatography/mass spectrometry (GC/MS) following solid phase extraction (SPE) and derivatization with bis(trimethyl-silyl) trifluoroacetamide (BSTFA). Examination found 330 microg/mL GHB in femoral blood and 221 ng/mg GHB in frontal cortex brain tissue, values higher than those typically reported in the literature. The hair shaft was negative for GHB whereas the plucked root bulbs with outer root sheath attached (2,221 ng/mg) and root bulbs after washing and removal of the outer root sheath (47 ng/mg) contained the drug. Our results are consistent with an acute single dose of GHB and, as the toxicology screen was negative for other drugs of abuse, emphasize the significant danger of this drug.     

The Antisaccade Task Performance Deficit and Specific CNV Abnormalities in Patients with Stereotyped Paraphilia and Schizophrenia,

Antisaccade task performance and mean amplitudes of slow cortical potentials (contingent negative variation—CNV) were investigated in 19 healthy volunteers, 16 schizophrenic patients (SP), and 12 patients with stereotyped form of paraphilia (PP). Compared with healthy subjects, schizophrenic and paraphilic patients committed significantly more erroneous saccades. The clear between‐group CNV differences were observed during the early CNV stage that is associated with cognitive aspects of preparatory set. In SP, as compared to controls, the significant decline of CNV amplitude was found at frontal‐central area. PP have demonstrated the lack of CNV over central and parietal regions, but their CNV amplitudes in frontal area did not differ from values of control group. Thus, two distinct types of CNV abnormalities have been found. The SP results have been interpreted as support for frontal dysfunction in schizophrenia. The disconnection between prefrontal cortex, sensorimotor cortex, and related subcortical structures is hypothesized in paraphilia group.     

Immunohistochemical research on brain stem injury: expression of heat shock protein 70 in rat brain after traumatic brain stem injury

The aim of this study was to determine the expression of heat shock protein 70 (HSP70) as molecular chaperones, following traumatic brain stem injury(TBSI). The expression of HSP70 was detected in respective brain regions of rats 1 h, 3 h, 6 h, 12 h, 24 h after brain stem stabbing wound. Numerous HSP70 immunoreactivity positive blood vessel endothelioid and glia cells were found in cerebral, cerebellar cortex and hippocampus after 1 h, with H.E stain unchanged. In the area near the focal of brain stem injury, HSP70 immunoreactivity positive neurons increased significantly after 1 h of injury, reaching the highest level after 3 h, much higher than in other areas. HSP70 immunoreactivity positive cells still existed after 24 h of injury. It was suggested that TBSI cause the defectiveness of protein structure in brain, and induce HSP70 proteolysis of denature protein including misfolding or aggregate. TBSI can be diagnosed regionally by the increasing of HSP70 immunoreactivity positive neurons in the focal of brain stems.     

Hypoglycemic brain damage

Hypoglycemia was long considered to kill neurons by depriving them of glucose. We now know that hypoglycemia kills neurons actively from without, rather than by starvation from within. Hypoglycemia only causes neuronal death when the EEG becomes flat. This usually occurs after glucose levels have fallen below 1 mM (18 mg/dl) for some period, depending on body glycogen reserves. At the time that abrupt brain energy failure occurs, the excitatory amino acid aspartate is massively released into the limited brain extracellular space and floods the excitatory amino acid receptors located on neuronal dendrites. Calcium fluxes occur and membrane breaks in the cell lead rapidly to neuronal necrosis. Significant neuronal necrosis occurs after 30 min of electrocerebral silence. Other neurochemical changes include energy depletion to roughly 25% of control, phospholipase and other enzyme activation, tissue alkalosis and a tendency for all cellular redox systems to shift towards oxidation. The neurochemistry of hypoglycemia thus differs markedly from ischemia. Hypoglycemia often differs from ischemia in its neuropathologic distribution, a phenomenon applicable in forensic practice. The border-zone distribution of global ischemia is not seen, necrosis of the dentate gyrus of the hippocampus can occur and a predilection for the superficial layers of the cortex is sometimes seen. Cerebellum and brainstem are universally spared in hypoglycemic brain damage. Hypoglycemia constitutes a unique metabolic brain insult.