Effect of toluene inhalation on astrocytes and neurotrophic factor in rat brain

Toluene, an abused substance in Japan, is a neurotoxic chemical that has been shown to have neurobehavioral and electrophysiological effects. In previous work, both acute and chronic effects of toluene on cells have been studied extensively. However, although glial cells are thought to play an important role in the survival of neurons in the brain, the effect of toluene on glial cell function has not yet been characterized. To elucidate this, the effect of toluene inhalation on astrocytes in rat brain was examined. Toluene exposure (1500 ppm for 4 h on 4-10 days) augmented glial fibrillary acidic protein (GFAP) immunoreactivity, particularly in the hippocampus and cerebellum. Quantitative analysis showed that toluene inhalation markedly enhanced GFAP expression in the hippocampus and cerebellum. In both regions, proliferating cell nuclear antigen (PCNA) showed no obvious changes, but glutamine synthetase (GS)-immunoreactive cells were markedly increased by toluene exposure. Thus, the elevation of GFAP expression was induced by astrocyte activation rather than by cell proliferation. If toluene exposure activates astrocytes, astrocytes may play a role in the neurophysiological changes observed in toluene intoxication. A neurotrophic factor, basic fibroblast growth factor (b-FGF) was observed immunohistochemically in the capillary vessel walls in the hippocampus and the cerebellum of toluene-intoxicated rats. Basic-FGF may have induced GFAP expression both in the hippocampus and the cerebellum. So, other neurotrophic factors may affect the difference of GFAP elevation between the hippocampus and the cerebellum. These differences may relate to neurobehavioral function of each brain part after toluene exposure.     

Lectin-histochemical detection of degenerative glycoconjugate deposits in human brain

Several lectins were used to study the localization of glycoconjugates in brain of elderly people and patients with Alzheimer type dementia (ATD) and Down's syndrome (DS). Five kinds of degenerated or deposited materials stained clearly by lectins specific to GalNAC, Gal, Fuc, and/or Man were recognized much in ATD and DS, less in elderly peoples, in addition to the binding of the lectins to neurons. (i) Round shape deposits called corpora amylacea (CA) which consisted of various sizes of round material, existed mainly on the surface of cerebral cortex and some in white matter of the brain. They were colored by Alcian blue (AB), Aldehyde fucsin (AF) and periodic acid shiff (PAS) and weakly by Hematoxylin (H), but not by Eosin (B). They showed clear reactivity with lectins specific to GalNAC, Gal, Fuc and Gal-GalNAC. (ii) Amorphous and variform amyloid deposits existed around blood vessels in the white matter were stained by thioflavin and lectins specific to GalNAC, Gal and Fuc, but not with Man specific lectins and PAS, AB, AF and HE. (iii) Another kind of amyloid deposits which showed a similar characteristic to the previous one and were recognized mainly in white matter and independent blood vessels. These deposits were stained by thioflavin but not by PAS, AB, AF and HE and showed good reactivity with lectins specific to GalNAC, Gal, Fuc, Gal-GalNAC, Gal-GIcNAc and Man. The reactivity with lectins specific to Gal, Fuc, and Man was seen in senile plaques (iv) and neurofibrillary tangles (v). Although at present we are unable to explain the origin of these deposits, it is clear from this study that the glycoconjugates form an integral part of the degeneration in the brain. The lectin staining with GS-I is useful in the forensic pathology to diagnose brain disorders at postmortem examination, since these lectin were able to detect five types of degeneration changes and/or deposits.     

Determination of sex from femora

The determination of sex from bones or bone fragments considerably contributes to identifying unknown bodies or skeletal remains. Due to temporal change and regional differences anthropometric standards have to be constantly renewed. The present study provides measurements of femoral dimensions in a contemporary German population and analyses sexual dimorphism by discriminant analysis. Maximum length (male: 46.4+/-2.4 cm, female: 43.4+/-2.4 cm), maximum midshaft diameter (male: 3.1+/-0.2 cm, female: 2.8+/-0.2 cm), condylar width (male: 8.4+/-1.0 cm, female: 7.7+/-0.5 cm), vertical head diameter (male: 4.9+/-0.3 cm, female: 4.4+/-0.3 cm), head circumference (male: 15.7+/-0.8 cm, female: 13.8+/-1.0 cm) and transverse head diameter (male: 4.9+/-0.3 cm, female: 4.3+/-0.3 cm) were measured in 170 femora, 100 from male (age: 16-92 years, mean: 60.8 years; body height: 153-190 cm, mean: 171 cm) and 70 from female (age: 20-96 years, mean: 72 years; body height: 146-175 cm, mean: 161 cm) individuals. In the discriminant analysis (leave-one-out-method) 67.7% of cases could be grouped correctly with the maximum length alone, 72.4% with the maximum midshaft diameter, 81.4% with the condylar width, 86.8% with the vertical head diameter, 87.7% with the head circumference and 89.6% with the transverse head diameter. The stepwise procedure with all head measurements showed that the results for the transverse head diameter could not be improved. With all measurements subjected to stepwise procedure 91.7% of cases could be classified correctly combining midshaft diameter and head circumference (D=3.012xmidshaft diameter in cm+0.780xhead circumference in cm 20.569).     

Screening and determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry

Benzodiazepines are one of the most widely prescribed drugs for the treatment of a wide spectrum of clinical disorders. They are used as anticonvulsants, anxiolytics, hypnotics or muscle relaxants with different duration of action. In this paper, a simple and sensitive method for the determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry (GC/MS) is described. The drugs spiked in whole blood were extracted with an Oasis HLB solid-phase extraction cartridge (Waters), which contains a copolymer designed to have a hydrophilic-lipophilic balance. GC/MS analysis was performed using a Shimadzu QP-5000 equipped with a BPX5 capillary column (15 mx0.32 mm I.D., film thickness 0.25 microm, SGE). Nineteen benzodiazepines and two thienodiazepines were well separated from each other on their SIM chromatograms and also on the TIC with the exception of oxazolam to cloxazolam separation. The blank extract from whole blood gave no peaks that interfered with all benzodiazepines and thienodiazepines on the chromatogram. The calibration curves for selected benzodiazepines with fludiazepam as an internal standard showed excellent linearity over the concentration range 5-500 ng/ml blood with a correlation coefficients of >0.995. The detection limits ranged from 0.2 to 20 ng/ml blood. The method is simple and sensitive for the determination of benzodiazepines in whole blood and seems to be useful in the practice of forensic science.     

Simultaneous determination of THC-COOH and THC-COOH-glucuronide in urine samples by LC/MS/MS

A fast method using liquid-liquid extraction and HPLC/tandem-mass spectrometry (LC/MS/MS) was developed for the simultaneous detection of 11-Nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid beta-glucuronide (THC-COOH-glucuronide) and 11-Nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine samples. This highly specific method, which combines chromatographic separation and MS/MS analysis, can be used for the confirmation of positive immunoassay results even without hydrolysis of the sample or derivatisation of extracts. Liquid-liquid extraction was optimised: with ethylacetate/diethylether (1:1, v/v) THC-COOH-glucuronide and THC-COOH could be extracted in one step. Molecular ions of the glucuronide (MH(+), m/z 521) and THC-COOH (MH(+), m/z 345) were generated using a PE/SCIEX turboionspray source in positive ionisation mode; specific fragmentation was performed in the collision cell of an API 365 triple-quadrupole mass spectrometer and yielded major fragments at m/z 345 (for THC-COOH-glucuronide) and m/z 327 as well as m/z 299 for both cannabinoids. Chromatographic separation was performed using a reversed-phase C8 column and gradient elution with 0.1% formic acid/1 mM ammonium formate and acetonitrile/0.1% formic acid. Retention times were 22.2 min for the glucuronide and 26.8 min for THC-COOH. After enzymatic hydrolysis of urine samples with beta-glucuronidase/arylsulfatase (37 degrees C, 5 h), THC-COOH-glucuronide was no longer detectable by LC/MS/MS in urine samples. However, the THC-COOH concentration was increased. For quantitation of THC-COOH, THC-COOH-D(3) was added to the urine samples as internal standard prior to analysis. From the difference of THC-COOH in the native urine and urine after enzymatic hydrolysis, molar concentration ratios of THC-COOH-glucuronide/THC-COOH in urine samples of cannabis users were determined and found to be between 1.3 and 4.5.     

Numerical density of delta-opioid receptor expressing neurons in the frontal cortex of drug-related fatalities

In animal experiment and in cell culture, chronic morphine treatment has been followed by a reduction as well as an increase of the delta-opioid receptor (OR) number. The present postmortem morphometric study of morphine-related fatalities of drug addicts (n=12, 22-35 years old, with blood unconjugated morphine levels from 27.1 to 407 ng/ml, m.v. 176.9 ng/ml) versus a non-addicted control group (n=13, 10-44 years old) is intended to examine whether chronic opiate exposure also affects the numerical density of deltaOR expressing neurons in the human neocortex (area 10 according to Brodmann (Vergleichende Lokalisationslehre der Grosshirnrinde (1909) Johann Ambrosius Barth, Leipzig)). For the immunohistochemical procedure, vibratome sections (100 microm) were incubated with a monoclonal antibody against the deltaOR diluted 1:100, and immunoreactive sites were visualized using an immunoperoxidase protocol. The numerical densities of OR expressing and Nissl-stained neurons were assessed morphometrically (camera lucida drawings). In both collectives, the anti deltaOR immunoreactivity was predominantly localized in pyramidal neurons of layers (L) II/III and V as well as in round and ovoid neurons of L VI. In the drug-related fatalities, the density of neurons expressing deltaOR protein amounted for 2515+/-240/mm(3), in the control group for 2616+/-204/mm(3), thus displaying no statistically significant difference. These findings go along with the binding behavior of opioid ligands in postmortem brains of heroin addicts revealing similar receptor densities and affinities in the control subjects and addicts.     

Methamphetamine-related fatalities in forensic autopsy during 5 years in the southern half of Osaka city and surrounding areas

To outline the recent features of methamphetamine-related fatalities from the medico-legal point of view, a retrospective investigation of forensic autopsy cases involving methamphetamine during a 5-year period (1994-1998) in the southern half of Osaka city and surrounding areas (about 1.57 million population) was undertaken. Among 646 autopsy cases, methamphetamine was detected in 15 victims (nine males, six females; 16-71 years of age; most frequently in males in their thirties). Primary scenes of fatal events were concentrated in the middle of the city. About half of them were transferred from emergency medical centers (survival time, up to 30 h). The cause and manner of death were: methamphetamine poisoning (n=4), homicide (n=4), accidental falls and aspiration from drug abuse (n=4), fire death (n=1), myocardial infarction (n=1), and cerebral hemorrhage (n=1) under drug influence. Usually injection scars and fresh puncture sites were found. Blood methamphetamine concentrations were 2.29-17.05 micromol/dl in the fatal poisoning, 0. 44-3.80 micromol/dl in deaths from other extrinsic causes (trauma), and 1.35-2.17 micromol/dl in cardio- and cerebrovascular strokes. Common complications were cardiomyopathy, cerebral perivasculitis and liver cirrhosis/interstitial hepatitis. Fatal and nonfatal methamphetamine poisonings are separately dealt with by the administrative medical examiner's office and in emergency medical centers. Tightly cooperative approaches of clinical and medico-legal experts are required for the effective social and medical management of drug abuse.     

Extraction and amplification of authentic DNA from ancient human remains

A total of 36 ancient human remains from 12 individuals (three tooth/bone samples each) and one sample each of three individuals from the newest time was typed in a blind study using the amelogenin sex test. Prior to this molecular sex determination the sex of the individual was determined morphologically. The success rate of the amelogenin amplifications was >90%. For every individual an unambiguous molecular sex typing result was obtained. Furthermore, the morphological and molecular sex determinations were in accordance with each other, giving evidence for the authenticity and ancient origin of the polymerase chain reaction (PCR) amplifications.     

DNA typing of human remains found in damp environments

DNA typing is often used to determine identity from human remains. The environment to which the material has been exposed, however, is crucial for the success of the investigation. Damp conditions in particular can cause a rapid degradation of DNA, even in bone and teeth, and thus reduce the chances of successful typing. Here, we present the results of investigations performed on four skulls that had been lying in a damp environment for periods ranging from almost 1 year to about 45 years. In none of these cases was DNA typing successful on bone or, where present, on teeth. Where remnants of brain tissue were used, however, complete STR typing was possible in one case, partial STR typing in another, and mtDNA sequencing could be carried out in three cases. These findings suggest that brain tissue is relatively resistant to putrefaction in damp environments and, unlike bone, appears to exhibit a certain degree of protection against DNA degradation.     

Application of mtDNA sequence analysis in forensic casework for the identification of human remains

In four forensic cases of unidentified skeletal remains investigated in the last year, we were able to attach three to missing persons. In one case we could show that the discovered bone sample did not fit to a missing child. The method for mitochondrial DNA analysis for the routine identification of skeletal remains was established in our institute by typing bone samples of defined age obtained from Frankfurt's cemetery. Reproducible results were obtained for bones up to 75 years old. For analysis the bone samples were pulverised to fine powder, decalcified and DNA was extracted. From the DNA we amplified a 404-bp fragment from HV-1 and a 379-bp fragment from HV-2 of the mtDNA control region. After sequencing of the PCR products, the results were compared to the Anderson reference sequence and to putative maternal relatives.