Sensitive and simultaneous quantification of 16 neurotransmitters and metabolites in murine microdialysate by fast liquid chromatography-tandem mass spectrometry.

The sensitive and simultaneous measurement of multiple neurotransmitters in microdialysate (MD) of freely moving mice is a prerequisite to study neurochemical imbalances in specific brain regions. The quantitative analysis of 16 neurotransmitters and metabolites, including serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), melatonin (ME), dopamine (DA), levodopa (l-DOPA), 3-methoxytyramine (3-MT), norepinephrine (NE), epinephrine (EP), homovanillinic acid (HVA), acetylcholine (ACh), deoxy carnitine (iso-ACh), choline (Ch), and É£-aminobutyric acid (GABA), adenosine (ADE), glutamine (Gln), and glutamic acid (Glu) was achieved within a chromatographic separation time of 6.5 min by the application of a biphenyl column coupled to an API-QTrap 5500 (AB SCIEX) mass spectrometer. Optimized chromatographic separation as well as high sensitivity allow the simultaneous analysis and precise quantification of 16 neurotransmitters and metabolites in artificial cerebrospinal fluid (CSF). Sample preparation procedure consisted of simply adding isotopically labeled internal standard solution to the microdialysis sample. The limits of detection in aCSF ranged from 0.025 pg (Ch) to 9.75 pg (Gln) and 85.5 pg (HVA) on column. Recoveries were between 83 and 111% for neurotransmitter concentrations from 0.6 to 45 ng/ml or 200 ng/ml with a mean intra-day and inter-day coefficient of variation of 7.6% and 11.2%, respectively. Basal extracellular concentrations of the following analytes: 5-HT, 5-HIAA, ME, DA, 3-MT, HVA, ACh, iso-ACh, Ch, GABA, ADE, Gln, and Glu were determined in the striatum of mice with a MD flow rate of 0.5 µl/min. This LC-MS/MS method leads to an accurate quantification of ACh and its isobaric structure iso-ACh, which were detected in the MD samples at ratios of 1:8.6. The main advantage of the high sensitivity is the miniaturization of the MD protocol with short sample collection times and volumes down to 5 µl, which makes this method suitable for pharmacological intervention and optogenetic studies to detect neurochemical changes in vivo.

Fast microglial activation after severe traumatic brain injuries.

Traumatic brain injury is among the leading causes of death in individuals under 45 years of age. However, since trauma mechanisms and survival times differ enormously, the exact mechanisms leading to the primary and secondary injury and eventually to death after traumatic brain injury (TBI) remain unclear. Several studies showed the versatile functions of microglia, the innate macrophages of the brain, following a TBI. Earlier being characterized as rather neurotoxic, neuroprotective capacities were recently demonstrated, therefore, making microglia one of the key players following TBI. Especially in cases with only short survival times, immediate microglial reactions are of great forensic interest in questions of wound age estimation. Using standardized immunohistochemical methods, we examined 8 cases which died causatively of TBI with survival times between minutes and 7 days and 5 control cases with cardiovascular failure as the cause of death to determine acute changes in microglial morphology and antigen expression after TBI. In this pilot study, we detected highly localized changes in microglial morphology already early after traumatic damage, e.g., activated microglia and phagocyted erythrocytes in the contusion areas in cases with minute survival. Furthermore, an altered antigen expression was observed with increasing trauma wound age, showing similar effects like earlier transcriptomic studies. There is minute data on the direct impact of shear forces on microglial morphology. We were able to show localization-depending effects on microglial morphology causing localized dystrophy and adjacent activation. While rodent studies are widespread, they fail to mimic the exact mechanisms in human TBI response. Therefore, more studies focusing on cadaveric samples need to follow to thoroughly define the mechanisms leading to cell destruction and eventually evaluate their forensic value.

Survival-time dependent increase in neuronal IL-6 and astroglial GFAP expression in fatally injured human brain tissue.

Knowledge on trauma survival time prior to death following a lethal traumatic brain injury (TBI) may be essential for legal purposes. Immunohistochemistry studies might allow to narrow down this survival interval. The biomarkers interleukin-6 (IL-6) and glial fibrillary acidic protein (GFAP) are well known in the clinical setting for their usability in TBI prediction. Here, both proteins were chosen in forensics to determine whether neuronal or glial expression in various brain regions may be associated with the cause of death and the survival time prior to death following TBI. IL-6 positive neurons, glial cells and GFAP positive astrocytes all concordantly increase with longer trauma survival time, with statistically significant changes being evident from three days post-TBI (p < 0.05) in the pericontusional zone, irrespective of its definite cortical localization. IL-6 staining in neurons increases significantly in the cerebellum after trauma, whereas increasing GFAP positivity is also detected in the cortex contralateral to the focal lesion. These systematic chronological changes in biomarkers of pericontusional neurons and glial cells allow for an estimation of trauma survival time. Higher numbers of IL-6 and GFAP-stained cells above threshold values in the pericontusional zone substantiate the existence of fatal traumatic changes in the brain with reasonable certainty.

Post-mortem in situ stability of serum markers of cerebral damage and acute phase response.

The aim of the given study was to test the in situ stability of biochemical markers of cerebral damage and acute phase response in the early post-mortem interval to assess their usability for forensic pathology. A monocentric, prospective study investigated post-mortem femoral venous blood samples at four time points obtained within 48 h post-mortem starting at the death of 20 deceased, using commercial immunoassays for the ten parameters: S100 calcium-binding protein B (S100B), glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), brain-derived neurotrophic factor (BDNF), interleukin-6 (IL-6), C-reactive protein (CRP), procalcitonin (PCT), ferritin, soluble tumor necrosis factor receptor type 1 (sTNFR1), and lactate dehydrogenase (LDH). Significant changes in serum levels were observed only later than 2 h after death for all markers. Inter-laboratory comparability was high, and intra-assay precision was sufficient for most markers. Most of the biomarker levels depended on the severity of hemolysis and lipemia but were robust against freeze-thaw cycles. Serum levels increased with longer post-mortem intervals for S100B, NSE, ferritin, sTNFR1, and LDH (for all p < 0.001) but decreased over this period for CRP (p = 0.089) and PCT (p < 0.001). Largely unchanged median values were found for GFAP (p = 0.139), BDNF (p = 0.106), and IL-6 (p = 0.094). Serum levels of CRP (p = 0.059) and LDH (p = 0.109) did not differ significantly between the final ante-mortem (resuscitation) and the first post-mortem sample (moment of death). Collecting the post-mortem blood sample as soon as possible will reduce the influence of post-mortem blood changes. Serum GFAP for detection of cerebral damage as well as serum IL-6 and CRP as proof of acute phase response seemed to be preferable due to their in situ stability in the first 2 days after death.

Frequency and intensity of pulmonary bone marrow and fat embolism due to manual or automated chest compressions during cardiopulmonary resuscitation.

Iatrogenic consequences of cardiopulmonary resuscitation (CPR) include sternal or rib fractures, pulmonary bone marrow embolisms (BME) and fat embolisms (FE). This report aimed to analyze the frequency and intensity of pulmonary BME and FE in fatal cases receiving final CPR efforts with the use of automated chest compression devices (ACCD) or manual chest compressions (mCC). The study cohort (all cardiac causes of death, no ante-mortem fractures) consisted of 15 cases for each group 'ACCD', 'mCC' and 'no CPR'. Lung tissue samples were retrieved and stained with hematoxylin eosin (n = 4 each) and Sudan III (n = 2 each). Evaluation was conducted microscopically for any existence of BME or FE, the frequency of BME-positive vessels, vessel size for BME and the graduation according to Falzi for FE. The data were compared statistically using non-parametric analyses. All groups were matched except for CPR duration (ACCD > mCC) but this time interval was linked to the existence of pulmonary BME (p = 0.031). Both entities occur in less than 25% of all cases following unsuccessful CPR. BME was only detectable in CPR cases, but was similar between ACCD and mCC cases for BME frequency (p = 0.666), BME intensity (p = 0.857) and the size of BME-affected pulmonary vessels (p = 0.075). If any, only mild pulmonary FE (grade I) was diagnosed without differences in the CPR method (p = 0.624). There was a significant correlation between existence of BME and FE (p = 0.043). Given the frequency, intensity and size of pulmonary BME and FE following CPR, these conditions may unlikely be considered as causative for death in case of initial survival but can be found in lower frequencies in autopsy histology.

Measurement of Cerebral Biomarkers Proving Traumatic Brain Injuries in Post-Mortem Body Fluids.

Until now, it is impossible to identify a fatal traumatic brain injury (TBI) before post-mortem radiological investigations or an autopsy take place. It would be preferable to have an additional diagnostic tool such as post-mortem biochemistry to get greater insight into the pathological pathways and survival times after sustaining TBI. Cerebrospinal fluid (CSF) and serum samples of 84 autopsy cases were collected from forensic autopsies with post-mortem intervals (PMI) of up to 148 h. The cases were categorized into a fatal TBI case group (n = 42) and non-TBI controls (n = 42). The values of glial fibrillary acidic protein (GFAP), brain-derived neurotrophic factor (BDNF), and neutrophil gelatinase-associated lipocalin (NGAL) were analyzed by means of quantitative chemiluminescent multiplex immunoassays. The main results indicate that the usage of liquid samples with good macroscopic quality is more relevant for meaningful biomarker analyses than the length of the PMI. All three proteins were shown to differentiate TBI fatalities from the controls in CSF. In serum, only GFAP could be shown to be able to identify TBI cases. This study is the first approach to measure the three proteins together in CSF and serum in autopsy cases. Determined threshold values may differentiate between fatal TBI and control cases. The presented results emphasize the possible use of post-mortem biochemistry as a supplemental tool in everyday forensic routine.

Acute phase response after fatal traumatic brain injury.

An inflammatory response occurring after fatal traumatic brain injury (TBI) initiates time-dependent cascades of acute phase response. This may offer the potential to monitor postmortem biomarker levels of several pro-inflammatory cytokines to gain information about the cause of death and the trauma survival time. Cerebrospinal fluid (CSF) and serum samples were collected from forensic autopsies of 95 adult cadavers after postmortem intervals up to 6 days. The cases were divided according to their cause of death into fatal TBI (n = 46) with different survival times and age- and gender-matching non-TBI fatalities as controls (n = 49). Quantitative marker levels of interleukin-6 (IL-6), ferritin, soluble tumor necrosis factor receptor type 1, C-reactive protein, and lactate dehydrogenase were analyzed using immunoassays. Standardized statistical tests were performed to differentiate causes of death and survival time of TBI cases. The CSF IL-6, ferritin, and LDH levels after TBI were significantly higher than those in the controls (p < 0.001). Only serum IL-6 values showed comparable differences (p < 0.05). Both CSF and serum ferritin levels were discriminative between early and delayed death after TBI (p < 0.05). There were partly distinctive correlations between marker levels in both fluids with rising values after longer survival. There were up to moderate correlation between the marker levels and the postmortem interval due to postmortem hemolysis. However, neither CSF nor serum level ranges were affected by the age or gender of the subjects. This study is the first to measure all five proteins systematically in postmortem trauma cases. Ferritin and IL-6 proved themselves to be interesting postmortem biomarkers to provide specific information on the injury pattern and the survival time of traumatic fatalities. Such forensic investigations could serve as inexpensive and fast laboratory tests.

Unusual planned complex suicide committed with a muzzle-loading pistol in combination with subsequent hanging.

In Germany, suicides by firearms are not very common in contrast to deaths by hanging and intoxications. The use of historical muzzle-loading firearms in the context of suicides is a rarity. Contact shots from muzzle loaders cause an unusual wound morphology with extensive soot soiling. We report the case of a 59-year-old man, who committed a planned complex suicide by shooting into his mouth with a replica percussion gun in combination with hanging. The gunshot injury showed strong explosive effects in the oral cavity with fractures of the facial bones and the skull associated with cerebral evisceration (so-called Krönlein shot). Due to the special constellation of the case with hanging immediately after the shot, external bleeding from the head injuries was only moderate. Therefore, the head injuries could be assessed and partially reconstructed already at the scene.

Survived ileocecal blowout from compressed air.

Industrial accidents with compressed air entering the gastro-intestinal tract often run fatally. The pressures usually over-exceed those used by medical applications such as colonoscopy and lead to vast injuries of the intestines with high mortality. The case described in this report is of a 26-year-old man who was harmed by compressed air that entered through the anus. He survived because of fast emergency operation. This case underlines necessity of explicit instruction considering hazards handling compressed air devices to maintain safety at work. Further, our observations support the hypothesis that the mucosa is the most elastic layer of the intestine wall.

An avoidable death in police custody?

An obviously inebriated 36-year-old man was found in a helpless condition on a pavement. When he was brought to a police station, a doctor certified the individual to be fit to be held in custody. He was unresponsive when the officers tried to wake him the next morning but was allowed to sleep on until he was found dead in his cell at mid day. An autopsy revealed as cause of death an epidural hematoma and cerebral contusion with fracture of the calvarium. The expert report stressed the point that the monitoring of the state of consciousness had been objectively insufficient throughout the entire time period, both in terms of frequency as well as in terms of the monitoring methods. However, neither the police officers involved nor the doctor, was subjected to prosecution mainly because of the high burden of proof required by German criminal law. The article will discuss conclusions for the avoidance of such deaths (such as training of police officers, legal regulations on monitoring procedures, etc).

Headspace solid-phase microextraction-gas chromatography-mass spectrometry determination of the characteristic flavourings menthone, isomenthone, neomenthol and menthol in serum samples with and without enzymatic cleavage to validate post-offence alcohol drinking claims.

A rapid HS-SPME-GC-MS (headspace solid-phase microextraction-gas chromatography-mass spectrometry) method has been developed for determination of menthone, isomenthone, neomenthol and menthol in serum samples with and without enzymatic cleavage. These flavour compounds are characteristic markers for consumption of peppermint liqueurs as well as certain digestif bitters, herbal and bitter liqueurs. This method enabled the detection of the four compounds with a limit of detection (LOD) of 2.1 ng mL(-1) (menthone and isomenthone), 2.8 ng mL(-1) (neomenthol) and 4.6 ng mL(-1) (menthol), and a limit of quantification (LOQ) of 3.1 ng mL(-1) (menthone and isomenthone), 4.2 ng mL(-1) (neomenthol) and 6.8 ng mL(-1) (menthol) in serum samples. The method shows good precision intraday (3.2-3.8%) and interday (5.8-6.9%) and a calibration curve determination coefficient (R(2)) of 0.990-0.996. Experiments were conducted with a volunteer, who consumed peppermint liqueur on three different days under controlled conditions. At defined intervals, blood samples were taken, and the concentration-time profiles for serum menthone, isomenthone, neomenthol and menthol, as free substances as well as glucuronides, were determined. Both menthol and neomenthol underwent a rapid phase II metabolism, but minor amounts of free substances were also detected. Menthone and isomenthone were rapidly metabolised and were found in lower concentrations and over a shorter time span than the other analytes. In blood samples taken from 100 drivers who claimed to have consumed peppermint liqueur prior to the blood sampling, menthone, isomenthone, neomenthol and menthol were detected in the serum as free substances in concentrations between 3.1 and 7.0 ng mL(-1) in eight cases (menthone), 3.1 and 11.3 ng mL(-1) in eight cases (isomenthone), 5.3 and 57.8 ng mL(-1) in nine cases (neomenthol) and 8.0 and 92.1 ng mL(-1) in nine cases (menthol). The sum values of free and conjugated substances ranged between 4.2 and 127.8 ng mL(-1) in 35 cases for neomenthol and 11.0 and 638.2 ng mL(-1) in 59 cases for menthol. Menthone and isomenthone were not conjugated. These test results confirmed that the analysis of characteristic beverage aroma compounds, such as menthone, isomenthone, neomenthol and menthol, can be used for specific verification of post-offence alcohol consumption claims.

Apoptosis and gene expression after TBI.

Proper CNS function depends on concerted expression of thousands of genes in a controlled and timely manner. Traumatic brain injury (TBI) in humans results in neuronal death and neurological dysfunction, which might be mediated by altered expression of several genes. By employing a CNS-specific GeneChip and real time polymerase chain reaction (PCR), the study analyzed the gene expression changes. The findings of necrosis and apoptosis can help to improve the wound age estimation after TBI in legal medicine.

Headspace solid-phase microextraction-gas chromatography-mass spectrometry for the quantitative determination of the characteristic flavouring agent eugenol in serum samples after enzymatic cleavage to validate post-offence alcohol drinking claims.

A rapid headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method has been developed for the determination of eugenol in serum samples after enzymatic cleavage. Eugenol is a characteristic marker for the consumption of certain alcoholic beverages including some digestif bitters and herbal liqueurs as well as wood-cask-aged spirits. This method enables the detection of eugenol with a limit of detection (LOD) of 3.2 ng/ml and a limit of quantification (LOQ) of 4.8 ng/ml in serum samples with excellent precision (5.3% intraday, 6.9% interday) and linearity (correlation coefficient R2=0.992). Our findings confirm that eugenol undergoes a rapid phase II metabolism as it occurs completely conjugated as eugenol glucuronide in serum. Free eugenol was not detectable in any of our samples, which necessitated enzymatic cleavage with beta-glucuronidase prior to HS-SPME sampling. In vivo experiments were conducted with a volunteer, who consumed a digestif bitter beverage on three different days under controlled conditions. At defined intervals, blood samples were taken from the subject. Using these blood samples, concentration/time profiles for serum eugenol glucuronide were determined. A rapid resorption leads to a peak eugenol glucuronide concentration directly after drinking (up to 1742 ng/ml if 78 mg of eugenol are ingested) followed by a decrease during the next 3h. Blood samples were also taken from 20 drivers claiming to have consumed drinks containing eugenol. In five of the samples, eugenol glucuronide was detected at serum concentrations ranging from 12.1 to 172.3 ng/ml. These test results, in particular, confirm that the analysis of volatile compounds can be useful in forensic toxicology for the verification of post-offence alcohol consumption claims.

Determination of anethole in serum samples by headspace solid-phase microextraction-gas chromatography-mass spectrometry for congener analysis.

A rapid headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method has been developed for the determination of anethole in serum samples. Anethole is a characteristic marker for the consumption of aniseed spirits. This method enabled the detection of anethole with a limit of detection (LoD) of 3.6 ng/ml and a limit of quantification (LoQ) of 5.3 ng/ml in serum samples with a good degree of precision intraday (2.8%) and interday (4.5%). Experiments were conducted with one volunteer, in which the subject consumed the alcoholic drink ouzo on 3 different days under controlled conditions. At defined intervals, blood samples were taken from the subject. Using these blood samples, the concentration-time profiles for anethole were determined. In blood samples taken from 50 drivers who claimed to have consumed drinks containing anethole (ouzo, raki and the German aniseed liqueur "Küstennebel") before the taking of the blood sample, anethole was detected in the serum in concentrations of between 5.4 and 17.6 ng/ml in 10 cases. This is the first report describing the qualitative and quantitative determination of a beverage-characteristic aroma compound - in this case anethole - in serum samples after consumption of alcoholic beverages.

Lethal manganese-cadmium intoxication. A case report.

A case of a lethal manganese-cadmium (Mn-Cd) intoxication is reported. The postmortem examination revealed a noticeable reddish-violet discolouration of the serous cutes of all body cavities, but there was no indication of any corrosive burns of the mucous membranes of the gastrointestinal tract. An Mn concentration of 899 micro g/l blood and a Cd concentration of 238 micro g/l blood were found in the deceased woman. These concentrations are higher than normal levels by a factor of about 100. A subacute or chronic manganese-cadmium absorption must be assumed.