Hideki Shojo

Genetic and histologic evidence implicates role of inflammation in traumatic brain injury-induced apoptosis in the rat cerebral cortex following moderate fluid percussion injury

Traumatic brain injury (TBI) causes massive brain damage. However, the secondary injury and temporal sequence of events with multiple mechanisms after the insult has not been elucidated. Here, we examined the occurrence of apoptosis and a causal relationship between inflammation and apoptosis in the TBI brain. Following a lateral moderate fluid percussion injury model of TBI in adult rats, microarray analyses detected apparent changes in the expression levels of apoptosis-related genes which revealed time-dependent expression patterns for 23 genes in the lateral cortex. The upregulated 23 genes included inflammatory cytokines such as interleukin 1 (IL-1) α, IL-1β, and tumor necrotic factor (TNF) which immediately increased at 3 h following the injury. Time-dependent gene expression profile analyses showed that apoptosis was subsequently induced following inflammation. These results taken together suggested changes in expression of apoptosis-related genes may be associated with inflammatory response. Accompanying this surge of cell death genes after TBI was a neurostructural pathologic hallmark of apoptosis characterized by leakage of cytochrome c into cytoplasm, DNA fragmentation and apoptotic cells in the lateral cortex of the impacted hemisphere. Caspase-3 positive cells in the TBI brain were initially sporadic after 3 h, but these apoptotic cells subsequently increased and populated the cerebral cortex at 6 and 12 h, and gradually reached a plateau by 48 h. Interestingly, the expression profile of CD68 macrophage labeled cells closely resembled that of apoptotic cells after TBI, including the role of inflammatory signaling pathway in the progression of apoptotic cell death. These results taken together suggest that TBI induced upregulation of apoptosis-related genes, concomitant with the detection of apoptotic brain pathology during the 3-48 h post-injury period, which may be likely mediated by inflammation. Therapies designed at abrogating apoptosis and/or inflammation may prove effective when initiated at this subacute TBI phase.

Changes in localization of synaptophysin following fluid percussion injury in the rat brain.

Traumatic brain injuries damage neurons and cause progressing dysfunctions of the brain. Synaptophysin (SYP), a major integral transmembrane protein of synaptic vesicles, provides a molecular marker for the synapse and serves as a functional marker of the brain. This study examined magnitude-dependent changes of SYP in the rat brain 2 days following low, moderate or high fluid percussion injuries and investigated time-dependent changes of SYP in the rat brain with moderate fluid percussion injury 2, 15 and 30 days after trauma using immunohistochemistry and Western blotting. SYP immunoreactivity increased in the lateral cortex and in the subcortical white matter, with increasing magnitude of injury and time after trauma. Increased SYP immunoreactivity was accompanied with degeneration of neuronal cell bodies, their processes and terminals as well as glial cell proliferations. Amounts of SYP measured by Western blotting remained unchanged in brains with moderate fluid percussion within 30 days after trauma. These findings indicate that trauma accumulates SYP at injured sites of neurons without changing SYP contents and that increased SYP immunoreactivity in the cerebral cortex following traumatic injury reflects an inhibition of synaptic vesicle transportation and dysfunction of synapses, thus providing a histological substrate for brain dysfunctions.

Accidental Fatal Hypothermia in Elderly People with Alzheimer's Disease

We report two forensic autopsy cases of fatal accidental hypothermia in an 89-year-old woman and a 76-year-old man who were found dead and unclothed. In both cases, Alzheimers disease (AD) was diagnosed by neuropathological examination. Wandering due to AD was determined as the cause of these accidents. Although paradoxical undressing in hypothermic victims is known to occur as a result of cold exposure, in our patients, undressing was attributed to dementia due to AD before they became hypothermic. These cases indicate that neuropathological examination is crucial to determining the cause of such accidents and that undressing is not always the result of hypothermia in elderly victims.

DJ-1 ameliorates ischemic cell death in vitro possibly via mitochondrial pathway.

DJ-1 is an important redox-reactive neuroprotective protein implicated in regulation of oxidative stress after ischemia. However the molecular mechanism, especially the mitochondrial function, by which DJ-1 protects neuronal cells in stroke remains to be elucidated. The aim of this study was to reveal whether DJ-1 translocates into the mitochondria in exerting neuroprotection against an in vitro model of stroke. Human neural progenitor cells (hNPCs) were initially exposed to oxygen-glucose deprivation and reperfusion injury, and thereafter, DJ-1 translocation was measured by immunocytochemistry and its secretion by hNPCs was detected by enzyme-linked immunosorbant assay (ELISA). Exposure of hNPCs to experimental stroke injury resulted in DJ-1 translocation into the mitochondria. Moreover, significant levels of DJ-1 protein were secreted by the injured hNPCs. Our findings revealed that DJ-1 principally participates in the early phase of stroke involving the mitochondrial pathway. DJ-1 was detected immediately after stroke and efficiently translocated into the mitochondria offering a new venue for developing treatment strategies against ischemic stroke.

Oxygen–Glucose‐Deprived Rat Primary Neural Cells Exhibit DJ‐1 Translocation into Healthy Mitochondria: A Potent Stroke Therapeutic Target

DJ‐1 is a key redox‐reactive neuroprotective protein implicated in regulation of oxidative stress after stroke. However, the molecular mechanism, especially the role of mitochondrial function, by which DJ‐1 protects neural cells in stroke remains to be elucidated. The aim of this study was to reveal whether DJ‐1 translocates into the mitochondria in exerting neuroprotection against oxidative stress. In particular, we examined DJ‐1 secretion from primary rat neural cells (PRNCs) exposed to experimental stroke.

A simple identification method of saliva by detecting Streptococcus salivarius using loop-mediated isothermal amplification.

Abstract:  We previously reported that detection of Streptococcus salivarius is feasible for proving the presence of saliva in a forensic sample. Here, a simple and rapid method for the detection of S. salivarius in forensic samples was developed that uses loop‐mediated isothermal amplification (LAMP). The LAMP primer set was designed using S. salivarius‐specific sequences of glucosyltransferase K. To simplify the procedure, the sample was prepared by boiling and mutanolysin treatment only, and the entire analytical process was completed within 2.5 h. The cut‐off value was set at 0.1 absorbance units, measured at 660 nm, upon termination of the reaction. S. salivarius was identified in all saliva samples, but was not detected in other body fluids or on the skin surface. Using this method, S. salivarius was successfully detected in various mock forensic samples. We therefore suggest that this approach is useful for the identification of saliva in forensic practice.

Changes in responsiveness of freshly isolated longitudinal muscle cells from rat uterus towards oxytocin during gestation: contractility and calcium signaling

Changes in responsiveness of freshly isolated longitudinal muscle cells from rat uterus to oxytocin during gestation were investigated through measuring contractility as well as intracellular free calcium concentration. We have demonstrated the pregnant stage-dependent contraction of freshly isolated myometrial cells in response to an extracellular hormone, oxytocin, in Ca2+-containing medium. The oxytocin effect appeared to be through oxytocin receptor since the effect could be blocked by a specific oxytocin antagonist. The magnitude of the contraction of the isolated cells in response to extracellular oxytocin was in the order of 21 day >> 18 day > 15 day pregnant rat longitudinal muscle cells. In a concentration dependent manner, oxytocin elicited a rapid increase in [Ca2+]i of longitudinal muscle cells isolated from different stages of the pregnant rat uterus, especially at the term of pregnancy. The time (4-5 s) required to reach a maximum increase in [Ca2+]i of the isolated longitudinal muscle cells in response to oxytocin was the shortest among all previously reported studies. The results also indicated that the freshly prepared longitudinal muscle cells maintained their functional calcium signaling system. The order of the responsiveness of the isolated longitudinal muscle cells to oxytocin was 21 day >> 18 day > 15 day pregnant rats in terms of rate, affinity and magnitude. Oxytocin appears to transmit its signal mainly through stimulating a voltage-dependent and/or receptor operated nonselective calcium channel. However, the possibility that a part of the oxytocin action occurs through stimulating the release of calcium from intracellular store sites of longitudinal muscle still remains.

Identification of feces by detection of Bacteroides genes.

In forensic science, the identification of feces is very important in a variety of crime investigations. However, no sensitive and simple fecal identification method using molecular biological techniques has been reported. Here, we focused on the fecal bacteria, Bacteroides uniformis, Bacteroides vulgatus and Bacteroides thetaiotaomicron, and developed a novel fecal identification method by detection of the gene sequences specific to these bacteria in various body (feces, blood, saliva, semen, urine, vaginal fluids and skin surfaces) and forensic (anal adhesions) specimens. Bacterial gene detection was performed by real-time PCR using a minor groove binding probe to amplify the RNA polymerase β-subunit gene of B. uniformis and B. vulgatus, and the α-1-6 mannanase gene of B. thetaiotaomicron. At least one of these bacteria was detected in the feces of 20 donors; the proportions of B. uniformis, B. vulgatus and B. thetaiotaomicron were 95, 85 and 60%, respectively. Bacteroides vulgatus was also detected in one of six vaginal fluid samples, but B. thetaiotaomicron and B. uniformis were not detected in body samples other than feces. Further, we applied this method to forensic specimens from 18 donors. Eighteen anal adhesions also contained at least one of three bacteria; B. uniformis, B. vulgatus and B. thetaiotaomicron were detected in 89, 78 and 56%, respectively, of the specimens. Thus, these bacteria were present at a high frequency in the fecal and forensic specimens, while either B. uniformis or B. vulgatus was detected in all samples. Therefore, B. uniformis and B. vulgatus represent more appropriate target species than B. thetaiotaomicron for the identification of fecal material. If B. vulgatus and/or B. uniformis are detected, it is likely that the sample contains feces. Taken together, our results suggest that the use of molecular biological techniques will aid the detection of feces in forensic practice, although it is possible that the samples contained both feces and vaginal fluid.

Epidemiological survey-based formulae to approximate incidence and prevalence of neurological disorders in the United States: a meta-analysis.

Background This study aims to create a convenient reference for both clinicians and researchers so that vis-à-vis comparisons between brain disorders can be made quickly and accurately. We report here the incidence and prevalence of the major adult-onset brain disorders in the United States using a meta-analysis approach. Material and Methods Epidemiological figures were collected from the most recent, reliable data available in the research literature. Population statistics were based on the most recent census from the US Census Bureau. Extrapolations were made only when necessary. The most current epidemiological studies for each disorder were chosen. All effort was made to use studies based on national cohorts. Studies reviewed were conducted between 1950 and 2009. The data of the leading studies for several neurological studies was compiled in order to obtain the most accurate extrapolations. Results were compared to commonly accepted values in order to evaluate validity. Results It was found that 6.75% of the American adult population is afflicted with brain disorders. This number was eclipsed by the 8.02% of Floridians with brain disorders, which is due to the large aged population residing in the state. Conclusions There was a noticeable lack of epidemiological data concerning adult-onset brain disorders. Since approximately 1 out of every 7 households is affected by brain disorders, increased research into this arena is warranted.

Inhibition mechanisms of hematophagous invertebrate compounds acting on the host blood coagulation and platelet aggregation pathways

To facilitate blood feeding, hematophagous invertebrates have evolved a sophisticated array of physiological compounds that counteract homeostatic systems and inflammatory reactions of the vertebrate host. For this reason, hematophagous invertebrates possess a variety of anticoagulation components that are inhibitors of coagulant factors or antagonists of the platelet receptor. The examination of kinetic data and the crystal structure analysis have exposed the inhibition mechanisms for many of these anticoagulant reagents. Here, we attempt to classify the antihemostatic molecules and to focus on the kinetic approaches that have been instrumental in defining these mechanisms.