A decrease in the tear secretion volume in a mouse model with ulcerative colitis.

PURPOSE: Dry eye syndrome is known to develop from several systemic inflammatory diseases. Although dry eye may develop due to extraintestinal complications of ulcerative colitis (UC), the pathogenesis is not well-known. This study aimed to investigate whether there was decrease in the tear secretion volume in a mice model with UC; the difference between the control and dextran sodium sulphate (DSS)-treated group was also determined. MATERIALS AND METHODS: This study included a mice model with UC induced by the oral administration of 5.0% DSS for 7 days. Following the DSS treatment, the tear volume was measured using the Schirmer's test. The colon and ocular tissues, including the lacrimal gland, were evaluated using histological and protein analyses. Additionally, tumour necrosis factor (TNF)-α and interleukin (IL)-6 in the plasma were determined. Differences between groups (DSS-treated versus control mice) were determined using Student's t-test. RESULTS: The tear volume in DSS-treated mice was decreased compared to that in the control mice. Plasma levels of TNF-α and IL-6 in DSS-treated mice was higher than that of control. Morphological change was observed with the invasion of the inflammatory cell in the lacrimal gland of DSS-treated mice. Terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL)-positive cells were increased in the lacrimal glands of DSS-treated mice compared with control group. The distribution of aquaporin-5 expressed in the lacrimal gland of DSS-treated mice was decreased compared to that in the control group. CONCLUSIONS: These findings suggest that a decrease in the tear volume in UC was associated with a functional decline in the inflamed lacrimal gland. This result therefore provides useful information that could contribute to the development of treatment approaches for dry eye associated with UC.

Circumstances and factors of sleep-related sudden infancy deaths in Japan.

BACKGROUND: Sudden unexpected death in infancy (SUDI) comprises both natural and unnatural causes of death. However, few epidemiological surveys have investigated SUDI in Japan. OBJECTIVE: This retrospective study was conducted to investigate the latest trends of circumstances and risk factors of SUDI cases in which collapse occurred during sleep. METHODS: Forensic pathology sections from eight universities participated in the selection of subjects from 2013 to 2018. Data obtained from the checklist form were analyzed based on information at postmortem. RESULTS: There were 259 SUDI cases consisting of 145 male infants and 114 female infants with a mean birth weight of 2888 ± 553 and 2750 ± 370 g, respectively. Deaths most frequently occurred among infants at 1 month of age (18%). According to population data as the control, the odds ratio (95% confidence interval) of mother's age ≤19 years was 11.1 (6.9-17.7) compared with ages 30-39. The odds ratio for the fourth- and later born infants was 5.2 (3.4-7.9) compared with the frequency of first-born infants. The most frequent time of day for discovery was between 7 and 8 o'clock, and the time difference from the last seen alive was a mean of 4.1 h. Co-sleeping was recorded for 61%, and the prone position was found for 40% of cases at discovery. Mother's smoking habit exhibited an odds ratio of 4.5 (2.9-5.8). CONCLUSION: This study confirmed the trends that have been observed for sudden infant death syndrome; particularly, very high odds ratios were evident for teenage mothers and later birth order in comparison with those in other developed countries. Neglect was suspected in some cases of the prolonged time to discovery of unreactive infants. To our knowledge, this is the first report of an extensive survey of SUDI during sleep in Japan.

Sudden death of a preschool child diagnosed by postmortem examination.

An autopsy case of sudden death due to pulmonary arterial hypertension (PAH) in a 5-year-old boy whose cause of death was not determined during autopsy, but was later determined by postmortem examination, is presented. The boy developed convulsions that subsequently stopped, but remained unconscious. He was transported to hospital by ambulance, but died soon after. The boy had been found to have right ventricular overload on ECG 2 weeks earlier. A plan had been made to consult a doctor for a specialist visit 2 months later. During autopsy, significant abnormalities or injuries were not observed on the body's external surface. Internal examination showed congested organs, and the blood remaining in the body was dark red with fluidity. The heart was significantly enlarged (146 g), with nearly equivalent thickness of the left and right ventricles, showing right ventricular hypertrophy. Obvious macroscopic abnormalities were not observed at the origin and main trunk of the pulmonary artery. The lungs were slightly swollen (right lung 100 g, left lung 95 g), severely congested, and edematous. A postmortem CT scan displayed some patchy shadows in both lungs; however, no significant abnormalities were detected. Histopathological examination suggested a diagnosis of PAH. Three genes (BMPR2, ALK1, and ENG) were tested, revealing a heterozygous insertion of five nucleotides, TTTCC, between nucleotides 2677 and 2678 within exon 12 of the BMPR2 gene. Therefore, the subject was considered to have had heritable PAH due to a BMPR2 gene mutation.

Autopsy findings for a case of acute gastric volvulus in a child.

Acute gastric volvulus resulting in abdominal compartment syndrome was determined to be the cause of death in a 4-year-old girl who presented with abdominal distension. At about 1AM on the day of her death, she was brought to our emergency medical center. Physical examination and plain abdominal X-ray revealed pronounced gastric dilatation. A decompression procedure was performed, followed by observation. She went into cardiopulmonary arrest around 1PM on the same day and died. Postmortem investigation, including an autopsy and computed tomography (CT), was performed to determine the cause of death. The findings included that the stomach was severely distended. Evidence was seen of mucosal hemorrhage in the gastric mucosa on the greater curvature side, which was thinned in places but without perforation. No necrosis of the gastric mucosa was observed; reversible changes were evident on histopathological examination. The postmortem CT images suggested that the pyloric region was positioned cranioventrally to the cardiac region. None of the findings indicated sudden blockage, and the cause of death was determined to be acute gastric volvulus resulting in abdominal compartment syndrome. The abnormal placement of the organs was difficult to determine based on physical examination alone; postmortem CT and careful examination were helpful in conducting the autopsy in this case.

A new molecular approach to help conclude drowning as a cause of death: simultaneous detection of eight bacterioplankton species using real-time PCR assays with TaqMan probes.

We developed a novel tool for concluding drowning as a cause of death. We designed nine primer pairs to detect representative freshwater or marine bacterioplankton (aquatic bacteria) and then used real-time PCR with TaqMan probes to rapidly and specifically detect them. We previously cultured the genus Aeromonas, which is a representative freshwater bacterial species, in blood samples from 94% of victims who drowned in freshwater and the genera Vibrio and/or Photobacterium that are representative marine bacteria in 88% of victims who drowned in seawater. Based on these results, we simultaneously detected eight species of bacterioplankton (Aeromonas hydrophila, A. salmonicida; Vibrio fischeri, V. harveyi, V. parahaemolyticus; Photobacterium damselae, P. leiognathi, P. phosphoreum) using three sets of triplex real-time PCR assays and TaqMan probes labelled with fluorophores (FAM, NED, Cy5). We assayed 266 specimens (109 blood, 157 tissues) from 43 victims, including 32 who had drowned in rivers, ditches, wells, sea or around estuaries. All lung samples of these 32 victims were TaqMan PCR-positive including the lung periphery into which water does not readily enter postmortem. On the other hand, findings in blood and/or closed organs (kidney or liver) were PCR-positive in 84% of the drowned victims (except for those who drowned in baths) although the conventional test detected diatoms in closed organs in only 44% of the victims. Thus, the results of the PCR assay reinforced those of diatom tests when only a few diatoms were detectable in organs due to the low density of diatoms in the water where they were found. Multiplex TaqMan PCR assays for bacterioplankton were rapid, less laborious and high-throughput as well as sensitive and specific. Therefore, these assays would be useful for routine forensic screening tests to estimate the amount and type of aspirated water.

Detection of diverse aquatic microbes in blood and organs of drowning victims: first metagenomic approach using high-throughput 454-pyrosequencing.

Current 454-pyrosequencing technology enables massive parallel sequencing. We used this technology to investigate the diversity of aquatic microbes in 14 specimens (blood and organs) of two drowning victims and in two water samples taken from the discovery sites. The 16S ribosomal RNA (rRNA) genes of microbes, which are often used to identify species (or genera), have nine highly variable regions (V1-V9), each of which is surrounded by conserved regions. Some parts within the conserved regions are common over domains of microbes, such as between bacteria and algae (16S rRNA genes on algal chloroplast genomes). We therefore simultaneously amplified the target regions (V7 and V8) of various microbes in the blood and organs of drowning victims using PCR with custom-designed primers that were based on the conserved regions. We then exhaustively analyzed the PCR products by pyrosequencing using the Genome Sequencer FLX Titanium system (Roche-454 Life Sciences). This approach identified a wide array of bacteria including cyanobacteria and algae including Bacillariophyceae (diatom), Cryptophyceae, Dictyochophyceae, Chrysophyceae and Trebouxiophyceae in the blood and organs of the victims and water at discovery sites. Our data further indicated that when conventional diatom testing of lungs yielded insufficient evidence of water aspiration, the detection of various exogenous microbes by 454-pyrosequencing is very useful to support a conclusion of death by drowning. To the best of our knowledge, this is the first attempt to use a new generation sequencer to investigate diverse aquatic microbes in the blood and closed organs of drowning victims.

Numbers, sizes, and types of diatoms around estuaries for a diatom test.

We collected 68 fresh, brackish, and seawater samples from various sites around the estuaries of 2 rivers at high and low tides. Seawater flowed approximately 2.4 (salinity, 2.2% at the site) and 1.2 km (1.8%) upstream of the estuaries, but the surface comprised essentially fresh water up to the mouth. Sites contained 69 to 22,200 diatoms/50 mL of water, and the numbers varied by depth and at sites separated by only approximately 1.2 km. Diatoms ranged from 2.8 to 429 µm (mean range, 16.1-59.2 µm) in size. Large pennate diatoms populated fresh water areas, and most sedimented before reaching the sea. Numbers of pennate diatoms of less than 20 µm were decreased in areas of seawater. Numbers of centric diatoms tended to increase nearer the sea, and seawater contained large centric diatoms. Brackish water containing large volumes of seawater was easily discriminated by assemblages of marine diatoms, unlike that containing a little seawater, because marine diatoms could be found in fresh water around estuaries. Tides and the nature of the river often altered diatomaceous assemblages at the same estuarial sites. Caution is recommended for forensic interpretation of aqueous media to deduce drowning sites.

Detection of marine and freshwater bacterioplankton in immersed victims: Post-mortem bacterial invasion does not readily occur.

We previously applied our method of detecting marine or freshwater bacterioplankton (bacteria) in the blood of immersed victims as a marker of drowning. However, we did not confirm the absence of post-mortem bacterial invasion during immersion. Here we examined the nature of bacterioplankton in blood samples from 21 immersed and 4 non-immersed cadavers. We found only freshwater bacterioplankton in the blood of two victims that were retrieved from the sea or an estuary inhabited by marine bacterioplankton even though one victim was highly putrefied. The results of diatom testing suggested that these two victims had drowned in fresh or brackish water with low salinity and then flowed out to the estuary or the sea. Two others were submerged in water, but representative bacterioplankton were undetectable in their blood although one victim was highly putrefied. Autopsy findings and the results of diatom tests did not indicate that the cause of death was drowning. As in previous studies, we identified freshwater bacterioplankton in the blood of seven other victims that had drowned in freshwater, marine bacterioplankton in the blood of four victims that had drowned in seawater and none in four victims found on land that had died by means other than drowning. Bacterioplankton in the blood of drowned victims appears to reflect the type of water aspirated and blood does not become easily contaminated with bacteria post-mortem even in decomposed bodies.

In vitro study of possible microbial indicators for drowning: Salinity and types of bacterioplankton proliferating in blood.

Numbers and types of bacterioplankton proliferating in blood samples mixed with water of various salinity levels were examined to determine the characteristics of species associated with salinity. Water samples (total n=88) were collected from the midstream of two rivers (freshwater; n=10; salinity <0.05%), from around their estuaries (areas of freshwater, n=20, salinity <0.05%; areas of brackish water, n=20, salinity <0.05-3.1%; areas of marine water beyond the mouths of the rivers, n=28, salinity 2.4-3.3%), and from the coast (areas of marine water; n=10; salinity 3.3-3.5%). Freshwater bacteria were identified in 41 of 42 blood samples mixed with water at ≤1.3% salinity, and the genus Aeromonas, which is universally distributed in freshwater environments, was predominant. Marine bacteria were identified in all of 46 blood samples mixed with water at ≥1.8% salinity, and most comprised the genera Vibrio and Photobacterium that are universally distributed in seawater environments. Aeromonas was undetectable in all blood samples mixed with brackish or sea water at ≥1.8% salinity although they are detectable even in seawater environments. Thus, the present results showed that bacterioplankton capable of proliferating in human blood reflects the salinity of water.

Autopsy of two frozen newborn infants discovered in a home freezer.

Two newborn infants (one male and one female) were discovered dead and frozen in a home freezer. Although thawing is expected to lead to changes that may hamper postmortem investigations, the victims could not be examined in the frozen state and were thus immersed in saline at 37 degrees C to completely thaw them over about 90 min. Autolysis and putrefaction were not evident, postmortem changes were slight, and the internal organs were soft, allowing a thorough examination, including an autopsy and a histological investigation. Autopsy showed that both infants were full-term at the time of death. Hydrostatic tests of the lung and stomach indicated that the infants had been born alive, and based on histological analyses of pulmonary alveoli and bronchioles, they had started breathing. Malformations, pathological findings, and signs of suspected asphyxia were absent, and we assume that the infants were very likely to have been murdered. However, the cause of death is not yet fully understood. Although a police investigation revealed that the infants were smothered by occlusion of the mouth and nose and then frozen, there were no injuries on the skin around the nose and mouth. Nonetheless, we concluded that none of the postmortem findings were in conflict with this suspected mechanism of death. As these infants had been kept frozen since their death and thawed to body temperature as rapidly as possible in saline at 37 degrees C, their bodies were well preserved, which was helpful for the postmortem investigation.

Freshwater bacterioplankton cultured from liver, kidney and lungs of a decomposed cadaver retrieved from a sandy seashore: possibility of drowning in a river and then floating out to sea.

A decomposed female body with an open abdomen and pleural cavity washed up on a beach after a powerful typhoon. Autopsy findings could not determine the cause of death because of leaching and putrefaction. Numbers and types of diatoms in organs overall, suggested the aspiration of fresh or brackish water with low salinity. However, this could not be confirmed because of contamination via the open cavities. We simultaneously investigated the presence of bacterioplankton in liver, kidney and lung homogenates using a modification of our reported bacteriological method. The freshwater bacterioplankton Plesiomonas shigelloides was identified in each of these organs, but marine bacterioplankton were undetectable despite the circumstances under which the body was discovered. The presence of freshwater bacterioplankton reinforced the results of the diatom test, and we concluded that this victim had died of drowning in fresh or brackish water with low salinity.

An autopsy case of chemical burns by hydrochloric acid.

A 34-year-old man was discovered by his coworkers in a tank filled with 35% (w/w) hydrochloric acid. Despite undergoing intensive treatment, he died one and a half days later. An autopsy revealed generalized high tensity, overall grayish brown skin color, heavy gastric submucosal hemorrhage and heavy pulmonary edema. We concluded that death was caused by burn shock due to wide, generalized chemical burn. Microscopic investigation of the burn in the area with grayish brown skin considered coagulation necrosis of full-thickness of the skin (third-degree or deep burn), revealed that the burn was judged to cover the partial thickness of the skin (second-degree or dermal burn). These findings suggest that chemical burn by hydrochloric acid results in a change of skin color due to chemical reaction so that the appearance of the chemical burn is more severe than the degree assigned by histological examination.

Detection of bacterioplankton in immersed cadavers using selective agar plates.

We measured bacterioplankton in blood from cadavers retrieved from the sea (n=12), near estuaries (n=4), rivers (fresh water, n=8) and from bathtubs (n=4) as well as from non-drowned victims (n=10) discovered near aquatic environments. Blood from 11 victims drowned in seawater developed bioluminescent and/or blue colonies (oxidase test positive) on selective media containing 2-4% NaCl. Homology analyses of the 16S rRNA gene showed that all of them were marine bacteria (genera: Photobacterium, Vibrio, Shewanella, Psychrobacter). Blood from all victims drowned in rivers generated blue colonies on plates containing 3%, but not 4% NaCl. Homology analyses showed that the blue colonies were generated from bacteria that inhabit fresh water (Aeromonas). None of the blood samples from victims that drowned in bathtubs generated bioluminescent and blue colonies. However, all cadavers contained bacteria that produced unstained colonies (Staphylococcus, Bacillus, Enterobacter, Escherichia, etc.). Among non-drowned victims, blood from two gave rise to blue colonies on plates containing < or =3% NaCl (Pseudomonas). Of the cadavers found near estuaries, bioluminescent and blue colonies developed from two of them on media containing 2-4% NaCl (Photobacterium, Vibrio, Listonella), but not from two others on plates containing 4% NaCl (at < or =3%; blue colonies, Aeromonas; unstained colonies, Citrobacter, Vagococcus, Proteus, Enterobacter). These results suggested that the presence of numerous bacterioplankton in immersed cadavers could support a conclusion of death by drowning.

Bioluminescent bacteria have potential as a marker of drowning in seawater: two immersed cadavers retrieved near estuaries.

We detected numerous bioluminescent bacteria in blood samples from two cadavers that had been immersed in estuarine environments. Autopsy, diatomaceous and toxicological findings indicated death by drowning, which agreed with environmental aspects and the findings of police investigations. Bioluminescent bacteria appeared in blood samples cultured on selective agar containing 2%, 3% and 4% NaCl after about 18h. Blood from the left side of the heart, the right side of the heart and the femoral vein generated 7.0 x 10(2), 2.0 x 10(4) and 8.0 x 10(2) cfu/ml of blood (case 1), and 1.8 x 10(4), 1.1 x 10(3) and 2.5 x 10(1) cfu/ml (case 2) of bioluminescent colonies, respectively, in agar containing 4% NaCl. Homologous analysis based on the 16S rRNA gene also identified the bioluminescent colonies as Vibrio fischeri and V. harveyi, which normally inhabit seawater. This simple assay might serve as an additional indicator to support a conclusion of death by drowning together with the diatom test.

Marine bacteria comprise a possible indicator of drowning in seawater.

To investigate the effectiveness of marine bacteria as a new marker of drowning in seawater, we determined the optimal conditions of media required to selectively detect marine bacteria and applied the technique to drowned cadavers. We incubated model blood samples (n=20 per group) mixed with seawater, river, tap or muddy water on agar plates (Todd Hewitt, TH; Marine 2216, M2216) and determined the NaCl concentration required to selectively detect marine bacteria. We also used TCBS agar plates without manipulation to isolate Vibrio spp. Among the culture media, TH agar was superior. Bioluminescent colonies were detected only in blood mixed with seawater. Blue colonies stained using the cytochrome oxidase test (COT), were detected in blood mixed with both sea and river water. However when the NaCl concentration was above 4%, COT stained colonies were detectable only in blood mixed with seawater. We subsequently used 2, 3 and 4% NaCl in TH and TCBS agar to examine blood from victims who had drowned in seawater (n=8) and in fresh water (n=7), as well as from victims who died near aquatic environments (non drowned; dry-land control, n=7). Bioluminescent colonies were detectable on 2-4% NaCl TH agar only from two victims that drowned in seawater. Bioluminescent colonies did not grow on TCBS agar. Blue colonies from all cadavers that had drowned in seawater (8/8) and in four of those that had drowned in fresh water (4/7) proliferated on TH agar containing 2% and/or 3% NaCl, but at 4% NaCl such colonies were detected only from cadavers that had drowned in seawater (8/8). Colonies from only one cadaver from seawater grew on TCBS agar. Furthermore, neither bioluminescent nor blue colonies were detected on TH agar containing 4% NaCl in samples from two cadavers found in an estuary (brackish water) who were thought to have been carried from areas of fresh water. Homologous analyses of the 16S rRNA gene revealed that the dominant colonies on TH agar containing 4% NaCl were marine bacteria (Photobacterium, Vibrio, Shewanella, Psychrobacter). Thus, proliferating bioluminescent and/or blue colonies detected in the blood of immersed cadavers using 4% NaCl TH agar, could help to establish drowning in seawater.

Effect of chronic ethanol administration on disposition of ethanol and its metabolites in rat.

We studied the effects of chronic alcohol intake on the disposition of alcohol and its metabolites in the rat. We used male Wistar rats for all of the experiments in this study. Using a pair-feeding process, rats were fed a liquid diet containing alcohol or without alcohol for 6 weeks. Ethanol solutions (0.5, 1.0, 1.5, and 2.0 g/kg body weight [BW]) were administered as a bolus, intravenously. We then measured blood ethanol and acetate concentrations. Simultaneous multiline fitting was performed using mean blood alcohol concentration (BAC)-time curves fitted to the one-compartment open model with parallel first-order and Michaelis-Menten elimination kinetics. At low doses (0.5, 1.0, and 1.5 g/kgBW), no differences were observed between the alcohol group and the control group with respect to ethanol elimination rate, area under the curve of ethanol (AUC(EtOH)), and mean residence time of ethanol (MRT(EtOH)). At higher doses (2.0 g/kgBW), ethanol elimination rate in the alcohol group was significantly higher than in the control group (P<.5%). These findings were also substantiated by corresponding changes in AUC(EtOH) and MRT(EtOH). At low doses, no differences were observed between the alcohol group and the control group with respect to plateau concentration of acetate (AcT) (concentration of steady state=C(ss)AcT), area under the curve of AcT (AUC(AcT)), and mean residence time of AcT (MRT(AcT)). However, at higher doses, although there were no differences in C(ss)AcT, both AUC(AcT) and MRT(AcT) were significantly lower in the alcohol group when compared to the control group (P<.5%). Chronic alcohol consumption increases ethanol oxidation and AcT metabolism in rats, as observed at high blood alcohol concentrations (BACs). These effects were observed at BACs of 3.5-4.5 mg/ml, and were not observed at lower doses. Thus, with general alcohol consumption, interindividual differences and intra-individual changes in alcohol metabolism may not take into account increased or accelerated metabolism due to alcohol tolerance.

Determination of endogenous testosterone in rat tissues following fetal alcohol exposure using HPLC with UV detection.

A novel method for quantitation of brain neurosteroid levels using HPLC with UV detection is described. In this simple and reliable method, testosterone from the brain and whole blood, and the internal standard, 17alpha-methyl testosterone, were extracted in 20% acetonitrile-phosphate buffer (pH 2.8), followed by solid phase extraction (SPE). The calibration curve was linear in concentration ranges from 0.1 to 10 ng from 0.2 g of tissue. We successfully applied this method to the analysis of endogenous testosterone in the male offspring of rats exposed to alcohol in utero. The concentration of testosterone at 21 post delivery in fetal alcohol exposure (FAE) group was significantly greater than the concentrations in either pair-fed or the ad libitum controls. These results support the usefulness of this method as a means of quantitating neurosteroids, and illustrate its applicability to fetal alcohol exposure.

Identification of DNA of human origin based on amplification of human-specific mitochondrial cytochrome b region.

Species-specific differences in a non-polymorphic region of the mitochondrial cytochrome b gene appear to be large enough to allow human-specific amplification of forensic DNA samples. We therefore developed a PCR-based method using newly designed primers to amplify a 157-bp portion of the human mitochondrial cytochrome b gene. The forward and reverse primers were designed to hybridize to regions of the human mitochondrial cytochrome b gene with sequences differing from those of chimpanzee by 26% (7 bp/27 bp) and 26% (6 bp/23 bp), respectively. Using this primer pair, we successfully amplified DNA extracted from blood samples of 48 healthy adults. All these human samples produced a single band of the expected size on agarose gel electrophoresis, and the sequence of the single band was shown to be identical to that of the target region (157 bp) by sequence analysis. On the other hand, no visible bands were amplified from DNA extracted from blood samples of animals including non-human primates (chimpanzee, gorilla, Japanese monkey, crab-eating monkey) and other species (cow, pig, dog, goat, rat, chicken and tuna). Thus, DNA producing a single band following PCR amplification using this primer pair can be reasonably interpreted as being of human origin. In addition, aged biological specimens comprising bloodstains, hair shafts and bones were successfully identified as being of human origin, illustrating the applicability of the present method to forensic specimens.