Perfusion with carbon monoxide does not affect extracellular glutamate in dialysates of the hippocampus of freely moving mice.

Carbon monoxide (CO) produces several neurological effects, including cognitive, mood, and behavioral disturbance. Glutamate is thought to play a particularly important role in learning and memory. Thus, the present study was aimed at investigating the local effect of CO on the glutamate level in the hippocampus of mice using in vivo reverse microdialysis. Mice were perfused with Ringer's solution (control) or CO (60-125 µM) in Ringer's solution into the hippocampus via microdialysis probe. Dialysate samples were collected every 20 min, and then analyzed with high-performance liquid chromatography coupled to an electrochemical detector. The result revealed that the perfusion with CO had no significant effect on glutamate levels (p = 0.316) as compared to the control group. This finding does not support a local CO rise as the cause of the increased glutamate level in the hippocampus of mice.

An autopsy case of death by combined use of benzodiazepines and diphenidine.

We present an autopsy case involving benzodiazepines and diphenidine. Quantitative toxicological analysis showed concentrations of 7-aminoflunitrazepam (a flunitrazepam metabolite), 7-aminonimetazepam (a nimetazepam metabolite), chlorpheniramine and diphenidine in femoral blood of 0.086 µg/ml, 0.027 µg/ml, 0.066 µg/ml, and 0.073 µg/ml, respectively. Death was attributed to combined toxicity due to the influence of multiple drug interactions.

Ethanol and Acetaldehyde After Intraperitoneal Administration to Aldh2-Knockout Mice-Reflection in Blood and Brain Levels.

This paper reports, for the first time, on the analysis of ethanol (EtOH) and acetaldehyde (AcH) concentrations in the blood and brains of Aldh2-knockout (Aldh2-KO) and C57B6/6J (WT) mice. Animals were administrated EtOH (1.0, 2.0 or 4.0 g/kg) or 4-methylpyrazole (4-MP, 82 mg/kg) plus AcH (50, 100 or 200 mg/kg) intraperitoneally. During the blood tests, samples from the orbital sinus of the eye were collected. During the brain tests, dialysates were collected every 5 min (equal to a 15 µl sample) from the striatum using in vivo brain microdialysis. Samples were collected at 5, 10, 15, 20, 25, 30 and 60 min intervals post-EtOH and -AcH injection, and then analyzed by head-space GC. In the EtOH groups, high AcH levels were found in the blood and brains of Aldh2-KO mice, while only small traces of AcH were seen in the blood and brains of WT mice. No significant differences in EtOH levels were observed between the WT and the Aldh2-KO mice for either the EtOH dose. EtOH concentrations in the brain were comparable to the EtOH concentrations in the blood, but the AcH concentrations in the brain were four to five times lower compared to the AcH concentrations in the blood. In the AcH groups, high AcH levels were found in both WT and Aldh2-KO mice. Levels reached a sharp peak at 5 min and then quickly declined for 60 min. Brain AcH concentrations were almost equal to the concentrations found in the blood, where the AcH concentrations were approximately two times higher in the Aldh2-KO mice than in the WT mice, both in the blood and the brain. Our results suggest that systemic EtOH and AcH administration can cause a greater increase in AcH accumulation in the blood and brains of Aldh2-KO mice, where EtOH concentrations in the Aldh2-KO mice were comparable to the EtOH concentrations in the WT mice. Furthermore, detection of EtOH and AcH in the blood and brain was found to be dose-dependent in both genotypes.

A fatal case of poisoning with ethanol and psychotropic drugs with putrefactive changes.

We present a fatal case involving poisoning with paroxetine, flunitrazepam, and ethanol, with putrefactive changes. Quantitative toxicological analysis showed that the concentrations of paroxetine and 7-aminoflunitrazepam, a metabolite of flunitrazepam, in the femoral blood were 0.28 µg/ml and 0.17 µg/ml, respectively. We also detected an ethanol level of 2.90 mg/ml and an n-propanol level of 0.10 mg/ml. We concluded that the cause of death was due to the interaction of paroxetine, flunitrazepam, and ethanol. The effects of putrefactive changes should be considered during forensic toxicological evaluation.

A color test for the convenient identification of an ingested surface activating agent.

Color tests are easy, simple and inexpensive methods for the qualitative identification of chemicals. A color test was applied to the stomach contents of a forensic autopsy case. The result of the test, using bromophenol blue reagent, indicated the ingestion of a commercial cleaning product containing a cationic surface activating agent. Our findings suggest that forensic investigators should consider the additives used in commercial chemical products, such as surface activating agents, when determining the cause of death.

Low-dose nicotine facilitates spatial memory in ApoE-knockout mice in the radial arm maze.

Here, we investigated the effects of nicotine on spatial memory in ApoE-knockout (ApoE-KO) and wild-type (WT) mice in a radial arm maze. Training occurred on three consecutive days and the test was performed on day 4, with one trial per day. Then on day 4, animals were administered nicotine (0.1, 0.25, 0.5, and 1.0 mg/kg) or the antagonist of nicotinic receptors (nAChRs) mecamylamine (MEC 2 mg/kg) alone or together with 0.1 mg/kg nicotine. The number of errors in the first eight choices was recorded. The results were that 0.1 mg/kg nicotine decreased errors in ApoE-KO mice, while 0.1 and 0.25 mg/kg nicotine reduced errors in WT mice, indicating that lower doses of nicotine elicit a memory improvement. In contrast, 1.0 mg/kg nicotine increased errors in WT mice, but not in ApoE-KO mice. MEC alone had no noticeable effect on errors in either strain of mice. However, co-administration of 0.1 mg/kg nicotine and MEC increased errors and reduced the effects of nicotine in WT mice, but not in ApoE-KO mice. Our study found a biphasic effect of nicotine in WT mice: it improves spatial memory at lower doses and impairs it at a higher dose. In ApoE-KO mice, nicotine improves memory at a low dose and has no effect at a higher dose, suggesting that the ApoE deficiency may influence the efficacy of nicotine. Moreover, a reversal of nicotinic effects with MEC was seen in WT mice, indicating the likelihood of the involvement of nAChRs in the spatial-memory response to nicotine.

Xylene; a useful marker for agricultural products ingestion.

Here we report about a case of malathion (an organophosphate insecticide) ingestion. Headspace analysis of stomach content provided useful information for screening of toxic chemicals. We should pay attention to solvents used in commercial industrial products.

Morphological evidence of an altered process of synaptic transcytosis in adult rats exposed to ethanol.

AIMS: The effects of ethanol exposure on synaptic structure were investigated in the nucleus of solitary tract (NST) in rats, using the horse-radish peroxidase (HRP) method. METHODS: Eight-week-old experimental rats were allowed free access to a liquid diet containing ethanol for 3 weeks, while controls were given an isocaloric diet. Some of the control and experimental animals were given an injection of wheat germ agglutinin conjugated with HRP (WGA-HRP) into the vagus nerve toward the end of the treatment period. After the treatment, the neuropil region of the NST was examined under an electron microscope. RESULTS: We observed that a few terminals were characterized by deep indentation of axodendritic membranes into the post-synaptic neurons. This appeared to be similar to that commonly seen in exocrine glands. Interestingly, the indented portion often contained various sizes of vacuoles and flattened cisternae. HRP-reaction product (RP) transported to terminals was recognized easily as an electron-dense lysosomal substance when lead citrate staining was omitted. Terminals containing HRP-RP also revealed quite a similar structure with indentation of axodendritic membranes as described earlier. The results are considered to confirm that terminals forming 'apocrine-like structures' observed in the ethanol-fed animals with no injection of WGA-HRP originate from afferent fibers of the vagus nerve. CONCLUSION: The present study suggests the possibility that the alteration of the synaptic structure induced by ethanol exposure can lead to the neuronal transcytosis of materials including proteins which is different from the normal vesicular exocytosis involved in chemical synaptic transmission.

Forensic toxicological implications of pleural effusion; an autopsy case of drug overdose.

UNLABELLED: A fatal poisoning case involving multiple psychotropic drugs is presented. Quantitative toxicological analysis showed that the concentrations of phenobarbital, promethazine, chlorpromazine, alprazolam and bromazepam in the femoral blood were 69.51µg/ml, 32.73µg/ml 24.76µg/ml, 0.04µg/ml and 0.31µg/ml respectively, and large amounts of drugs were also detected in the stomach contents. We concluded that the cause of death was mainly due to overdose of phenobarbital, promethazine and chlorpromazine by massive ingestion, and we also discuss the value of pleural effusions as an alternative specimen for toxicological examination. KEYWORDS: multiple drug - poisoning - liquid chromatography mass spectrometry - phenobarbital - pleural effusion.

A case of drowning whilst under the influence of brotizolam, flunitrazepam and ethanol.

A case of drowning involving brotizolam, flunitrazepam and ethanol ingestion was presented. Quantitative toxicological analysis showed that the concentrations of brotizolam, 7-aminoflunitrazepam (a metabolite of flunitrazepam) and ethanol in the femoral blood were 0.025 microg/ml, 0.094 microg/ml and 0.29 mg/ml, respectively, and these drugs were also detected in the stomach contents. We concluded that the cause of death was drowning whilst under the influence of combined use of brotizolam, flunitrazepam and ethanol.

An autopsy case of multiple psychotropic drug poisoning.

A fatal poisoning case involving etizolam, phenobarbital, promethazine and chlorpromazine is presented. Quantitative toxicological analysis showed that the concentrations of etizolam, phenobarbital, promethazine and chlorpromazine in the femoral blood were 86 ng/ml, 5082 microg/ml, 0.107 microg/ml and 0.144 microg/ml, respectively, and large amounts of drugs were also detected in the stomach contents. We conclude that the cause of death was due to the interaction of multiple psychotropic drugs.

Effects of systemic nicotine, alcohol or their combination on cholinergic markers in the frontal cortex and hippocampus of rat.

Acute alcohol (Alc) intoxication has been shown to decrease choline acetyltransferase (ChAT) in the rat brain. The present study extends that finding by examining the effects of nicotine (Nic), Alc, and their combination on ChAT and acetylcholinesterase (AChE) in the frontal cortex and hippocampus of rat. The samples were collected at 30 and 120 min after intraperitoneal administration of saline (0.9%, control), Nic (1 mg/kg), Alc (1 g/kg), and Nic + Alc and analyzed by RT-PCR, Western blot and colorimetry. Alc alone considerably reduced ChAT mRNA expression, whereas Nic alone decreased AChE mRNA expression. In contrast, Nic + Alc exposure had resulted in no significant change in the parameters. These findings are consistent with the results of the Western blot and AChE activity analysis. The results, therefore, indicate that Nic and Alc alone may interact with the central cholinergic system. This interactive effect may contribute to a frequent association of tobacco and Alc consumption.

An autopsy case of butane gas abuse.

A case of fatal butane gas poisoning in a young female is presented. Quantitative toxicological analysis showed that the concentration of butane in the femoral blood was 6.8 microl/ml, and isobutane and propane were also identified. Severe congestion of the lungs and deposition of lipofuscin in the myocardium were also observed. We concluded that the cause of death of the victim was due to cardiac arrhythmia induced by the butane gas abuse.

An autopsy case of carbamazepine poisoning.

We present a case of fatal carbamazepine poisoning. Quantitative analysis of carbamazepine using high performance liquid chromatography, revealed that the concentrations of carbamazepine were 50.2 microg/ml in the femoral venous blood and 60.3 microg/ml in the heart blood, respectively, and many unabsorbed tablets were also observed in the stomach contents. We concluded that the cause of death was due to carbamazepine overdose.

High ethanol and acetaldehyde decrease extracellular glutamate in the frontal cortex of freely moving mice.

Our recent study demonstrated that local perfusion of ethanol (EtOH) and acetaldehyde (AcH) into the hippocampus via microdialysis decreased extracellular glutamate; however, it is not clear whether this effect occurs in the frontal cortex. To address this issue, we investigated the effects of local perfusion of EtOH and AcH on extracellular glutamate in the frontal cortex of Aldh2-knockout (Aldh2-KO) and C57BL/6 N [wild-type (WT)] mice. Dialysates were collected every 20 minutes, and extracellular glutamate was measured using HPLC coupled with electrochemical detector. We found local perfusion of 200 and 500 mM EtOH into the frontal cortex of WT and Aldh2-KO mice produced significant decreases in extracellular glutamate levels (P < 0.05). A dose of 500 mM EtOH induced a greater decrease in Aldh2-KO mice (P < 0.05) than in WT mice, indicating the action of AcH. Similarly, perfusion of 200 and 500 µM AcH decreased glutamate in the frontal cortex of Aldh2-KO mice (P < 0.05), but this decrease was not seen in WT mice at any AcH dose, due to the subsequent oxidation of AcH by mitochondrial aldehyde dehydrogenase 2. A low dose of EtOH (100 mM) or AcH (100 µM) had no effect on glutamate. These results showed that high doses of EtOH and AcH induces a significant decrease in extracellular glutamate in the frontal cortex of mice, replicating previous findings and providing further evidence that reduced glutamate is likely to be involved in the depressant effects of EtOH.

High Ethanol and Acetaldehyde Inhibit Glutamatergic Transmission in the Hippocampus of Aldh2-Knockout and C57BL/6N Mice: an In Vivo and Ex Vivo Analysis.

We aimed to investigate whether ethanol (EtOH) and acetaldehyde (AcH) can affect glutamate and its receptors GluN1 and GluA1 in the hippocampus of Aldh2-knockout (Aldh2-KO) and C57BL/6N (wild-type (WT)) mice. To do this, we first examined the effect of local administration of EtOH (100 mM, 200 mM, and 500 mM) and AcH (100 µM, 200 µM, and 500 µM) on extracellular glutamate levels in freely moving mice. Retrodialysis of 200 mM and 500 mM EtOH into the hippocampus of WT and Aldh2-KO mice produced significant decreases in extracellular glutamate levels (p < 0.05). A dose of 500 mM EtOH induced a greater decrease in Aldh2-KO mice (p < 0.05) than in WT mice, indicating the action of AcH. Similarly, perfusion of 200 µM and 500 µM AcH decreased glutamate in Aldh2-KO mice (p < 0.05), but this decrease was not seen in WT mice at any AcH dose. Second, we tested whether the EtOH- and AcH-induced decrease in glutamate was associated with decreases in GluN1 and GluA1 expression, as measured by real-time PCR and Western blot. We found a significant decrease in GluN1 (p < 0.05) and GluA1 (p < 0.05) subunits after a high dose of EtOH (4.0 g/kg) and AcH (200 mg/kg) in WT mice. However, a 2.0 g/kg dose of EtOH did not produce a consistent decrease in GluN1 or GluA1 between messenger RNA and protein. In Aldh2-KO mice, all three doses of EtOH (1.0 g/kg, 2.0 g/kg, and 4.0 g/kg) and AcH (50 mg/kg, 100 mg/kg, and 200 mg/kg) decreased GluN1 expression (p < 0.05), while moderate-to-high doses of EtOH (2.0 g/kg and 4.0 g/kg) and AcH (100 mg/kg and 200 mg/kg) decreased GluA1 expression (p < 0.05). Together, these in vivo and ex vivo data suggest that EtOH and AcH decrease extracellular glutamate in the hippocampus of mice with a concomitant decrease in GluN1 and GluA1 subunits, but these effects require relatively high concentrations and may, therefore, explain the consequences of EtOH intoxication.

Analysis of the tracheal contents using headspace gas chromatography-mass spectrometry to screen for accelerant use.

We describe here the usefulness of analysis of tracheal contents in a case of death by fire, which revealed that the deceased had used the accelerants. The analysis of tracheal contents provides useful information for the determination of the circumstances of the scene.

COA-Cl induces dopamine release and tyrosine hydroxylase phosphorylation: In vivo reverse microdialysis and in vitro analysis.

We found that local perfusion of COA-Cl (0.1, 0.4, or 1.0 mM) into the dorsal striatum of living mice produced a significant and dose-dependent increase in extracellular DA levels, with the highest dose of 1.0 mM COA-Cl producing an approximately 5-fold increase in DA. Consistent with in vivo findings, 0.1 and 0.2 mM COA-Cl significantly and dose-dependently enhanced DA release 3.0 to 5.0-fold in PC12 cells, an in vitro model of DA-responsive neurons. Interestingly, the increase in striatal DA levels by COA-Cl in vivo was similar in magnitude to that observed in PC12 cells. Treatment with 0.1 mM COA-Cl significantly increased both Ser31 and Ser40 phosphorylation of tyrosine hydroxylase (TH) in PC12 cells, and Ser40 phosphorylation in iCell neurons, without altering total TH protein levels. Further, we examined whether COA-Cl could stimulate neurite outgrowth in PC12 cells and iCell neurons and found that COA-Cl significantly induced neurite outgrowth in both cell lines. Our results provide the first evidence that COA-Cl can stimulate dose-dependent DA release and activation of TH phosphorylation, suggesting that COA-Cl may be a promising therapeutic candidate for the treatment of neurological dysfunction associated with low DA.

Medico-legal investigation of chicken fat clot in forensic cases: immunohistochemical and retrospective studies.

Chicken fat clot (CFC), a fibrin-like substance, is sometimes found in the heart and large blood vessels in some autopsy cases. Reports of detailed histological findings of CFC are scant. We therefore examined CFC histologically in 53 autopsy cases and its correlation with ante-mortem or post-mortem evidence. We found three microscopic patterns of CFC: (1) wavelike fibrin fibers (WFF), (2) short fibrin fibers (SFF), and (3) short fibrin fibers mixed with wavelike fibrin fibers (SFF+WFF). WFF were found in the cases that survived less than 3 h after poisoning, burns, asphyxia, intracerebral hemorrhage, etc. SFF were found in the cases that survived more than 1 day after malignant neoplasms and acute or chronic inflammatory diseases, etc. SFF+WFF were found in the cases that died of inflammatory diseases, chronic heart failure, hemorrhagic shock, drowning, etc. About two-thirds of the SFF+WFF cases survived more than 1 day, with the rest surviving less than that. Our study confirmed three CFC patterns and their relation with survival interval. Therefore, these findings can be used as an index of the survival interval of a few acute and most chronic medico-legal death cases.

An autopsy case of poisoning with ethanol and psychotropic drugs.

A case of fatal poisoning involving ethanol with psychotropic drugs is presented. Quantitative toxicological analysis showed that the concentrations of ethanol, amoxapine and phenobarbital in the femoral blood were 2.86 mg/ml, 0.41 microg/ml and 6.80 microg/ml, respectively. We concluded that the cause of death was due to the combination use of ethanol, amoxapine and phenobarbital.