Kazuhiko Kibayashi

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.

Facial soft tissue thickness differences among three skeletal classes in Japanese population

Facial reconstruction is used in forensic anthropology to recreate the face from unknown human skeletal remains, and to elucidate the antemortem facial appearance. This requires accurate assessment of the skull (age, sex, ancestry, etc.) and thickness data. However, additional information is required to reconstruct the face as the information obtained from the skull is limited. Here, we aimed to examine the information from the skull that is required for accurate facial reconstruction. The human facial profile is classified into 3 shapes: straight, convex, and concave. These facial profiles facilitate recognition of individuals. The skeletal classes used in orthodontics are classified according to these 3 facial types. We have previously reported the differences between Japanese females. In the present study, we applied this classification for facial tissue measurement, compared the differences in tissue depth of each skeletal class for both sexes in the Japanese population, and elucidated the differences between the skeletal classes.

Changes in dopamine transporter expression in the midbrain following traumatic brain injury: an immunohistochemical and in situ hybridization study in a mouse model.

Abstract Objectives: An association has been suggested between trauma and neurological degenerative diseases. Magnetic resonance imaging has revealed that traumatic brain injury (TBI) can cause primary lesions in the midbrain including the substantia nigra (SN). Dopamine transporter (DAT) is mainly expressed in the SN, ventral tegmental area (VTA), and retrorubral field (RRF) of the ventral midbrain. Previous western blot studies have examined DAT levels in the rat frontal cortex and striatum after a controlled cortical impact (CCI); however, no study has comprehensively examined DAT expression in the midbrain following TBI in an animal model. Methods: We used immunohistochemistry and in situ hybridization to examine the time-dependent changes in the expression of DAT in the midbrain during the first 14 days after TBI in a mouse CCI model. Results: The expression of DAT protein in the RRF on the side ipsilateral to the site of injury decreased in 14 days after injury. Dopamine transporter mRNA expression in the RRF on the ipsilateral side decreased in 1, 7, and 14 days and increased in 4 days after injury. Discussion: These findings indicated that TBI induced changes in DAT expression in the RRF. Because the DAT pumps dopamine (DA) out of the synapse back into the cytosol and maintains DA homeostasis, the decreased expression of DAT after TBI may result in decreased DA neurotransmission in the brain.

Mild hypothermia facilitates the expression of cold-inducible RNA-binding protein and heat shock protein 70.1 in mouse brain

An appropriate thermal control system is essential for maintaining brain homeostasis. Hypothermia is a decrease in core body temperature that occurs when the thermoregulatory responses of homeothermic animals are impaired by environmental and situational influences, such as cold ambience and anesthesia. In recent years, hypothermia has been used for medical treatment, i.e., therapeutic hypothermia, for patients with stroke, traumatic brain injury, and heart surgery. However, the target molecules acting during hypothermia have not been identified. To understand the molecular mechanisms, we generated a mouse model of mild hypothermia (1°C-2°C below normal), and analyzed the expression of several genes. After mice were exposed to cold for 24 and 48 h, their rectal temperature reached 33°C-35°C. Then, using real-time quantitative PCR, we analyzed the mRNA expression levels of c-fos, cold-inducible RNA-binding protein (CIRP), heat shock protein (hsp) 70.1, oxytocin, and representative inflammatory cytokines, i.e., tumor necrosis factor (TNF)-α and interleukin (IL)-6 in target organs. Importantly, we found that the expression levels of CIRP and hsp70.1 were elevated in the olfactory bulb within 48 h. In the hypothalamus, CIRP expression levels increased and were followed by an increase in hsp70.1 expression. Meanwhile, TNF-α and IL-6 expression decreased gradually over 24 and 48 h in the olfactory bulb and hypothalamus. These specific expression profiles, i.e., enhanced CIRP and hsp70.1 expression and depressed cytokine expression, suggest that they could regulate apoptosis related to the cytokine signaling.

Changes in the Brain after Intracerebral Implantation of a Lead Pellet in the Rat

In gunshot wounds to the head, the bullet containing neurotoxic lead may remain in the brain after trauma, and brain damage is therefore anticipated. We developed an animal model incorporating a lead ball implanted in the brain, or a glass ball as a control, and analyzed histological and biochemical changes in the brain for 28 days after surgery. The concentration of lead in the brain increased with time after implantation of the lead ball, while lead was not detected in brains implanted with a glass ball. A number of changes were noted following implantation of the lead ball. The number of neutrophils increased significantly at 7-28 days, while the number of macrophages also increased significantly with time. The number of neurons was significantly decreased by 28 days, and apoptotic cells were identified at 14-28 days. Expression of genes encoding N-methyl-d-aspartate receptors, which are related to cognitive function and help regulate apoptosis, was significantly suppressed at 7-21 days after implantation. Expression of metallothionein protein increased significantly with time. Metallothionein detoxifies lead and its overexpression is a known method of protection against lead neurotoxicity. These findings indicate that intracerebral lead induces neuronal degeneration that is attributable in part to the inflammation and apoptosis caused by leached lead. Although the severity of the brain damage primarily depends on the destructive effect of the moving bullet, the results of this study indicate that lead leaching from the residual bullet also damages the brain.

A rat model of changes in dural mast cells and brain histamine receptor H3 expression following traumatic brain injury

Mast cells can secrete histamine in response to extrinsic stimuli. Histamine plays a role in the development of brain edema and can induce histamine receptor H3 (HRH3) expression in the brain to provide protective feedback effects against histamine neurotoxicity. We investigated time-dependent changes in dural mast cell numbers and HRH3 expression in the brain for one to 14 days after traumatic brain injury in a controlled cortical impact model in the rat. The number of tryptase-immunoreactive dural mast cells at the site of impact was significantly decreased one and four days after the injury. Furthermore, immunoreactivity and messenger RNA (mRNA) expression of HRH3 at the underlying cortical contusion site were significantly increased one and four days after the injury. These data suggest that histamine released from degranulated unstainable mast cells induces a transient increase in presynaptic autoinhibitory HRH3 immunoreactivity and mRNA expression as a mechanism to counteract histamine neurotoxicity.

A simple ABO genotyping by PCR using sequence-specific primers with mismatched nucleotides.

In forensics, the specific ABO blood group is often determined by analyzing the ABO gene. Among various methods used, PCR employing sequence-specific primers (PCR-SSP) is simpler than other methods for ABO typing. When performing the PCR-SSP, the pseudo-positive signals often lead to errors in ABO typing. We introduced mismatched nucleotides at the second and the third positions from the 3-end of the primers for the PCR-SSP method and examined whether reliable typing could be achieved by suppressing pseudo-positive signals. Genomic DNA was extracted from nail clippings of 27 volunteers, and the ABO gene was examined with PCR-SSP employing primers with and without mismatched nucleotides. The ABO blood group of the nail clippings was also analyzed serologically, and these results were compared with those obtained using PCR-SSP. When mismatched primers were employed for amplification, the results of the ABO typing matched with those obtained by the serological method. When primers without mismatched nucleotides were used for PCR-SSP, pseudo-positive signals were observed. Thus our method may be used for achieving more reliable ABO typing.

Evaluation of the morphological changes of gastric mucosa induced by a low concentration of acetic acid using a rat model

Oral ingestion of concentrated acetic acid causes corrosive injury of the gastrointestinal tract. To assess the effects of a low concentration of acetic acid on gastric mucosa, we examined the gastric mucosal changes in rats at 1 and 3 days after the injection of 5% or 25% acetic acid into the gastric lumen. The area of the gastric ulcerative lesions in the 25% acetic acid group was significantly larger than that in the 5% acetic acid group. The lesion area was reduced significantly at 3 days after injection in the 5% acetic acid group, whereas no significant difference in lesion area was observed at 1 and 3 days in the 25% acetic acid group. Histologically, corrosive necrosis was limited to the mucosal layer in the 5% acetic acid group, whereas necrosis extended throughout the gastric wall in the 25% acetic acid group. At 3 days post-injection, the 25% acetic acid group showed widespread persistent inflammation, whereas the 5% acetic acid group showed widespread appearance of fibroblasts indicative of a healing process. These results indicate that a low concentration of acetic acid damages the gastric mucosa and that the degree of mucosal damage depends on the concentration of acetic acid.

Pilot study to establish a nasal tip prediction method from unknown human skeletal remains for facial reconstruction and skull photo superimposition as applied to a Japanese male populations

Skull-photo superimposition is a technique used to identify the relationship between the skull and a photograph of a target person: and facial reconstruction reproduces antemortem facial features from an unknown human skull, or identifies the facial features of unknown human skeletal remains. These techniques are based on soft tissue thickness and the relationships between soft tissue and the skull, i.e., the position of the ear and external acoustic meatus, pupil and orbit, nose and nasal aperture, and lips and teeth. However, the ear and nose region are relatively difficult to identify because of their structure, as the soft tissues of these regions are lined with cartilage. We attempted to establish a more accurate method to determine the position of the nasal tip from the skull. We measured the height of the maxilla and mid-lower facial region in 55 Japanese men and generated a regression equation from the collected data. We obtained a result that was 2.0±0.99mm (mean±SD) distant from the true nasal tip, when applied to a validation set consisting of another 12 Japanese men.

Traumatic brain injury decreases serotonin transporter expression in the rat cerebrum.

Objectives: An association has been postulated between traumatic brain injury (TBI) and depression. The serotonin transporter (SERT) regulates the concentration of serotonin in the synaptic cleft and represents a molecular target for antidepressants. We hypothesized that SERT expression in the brain changes following TBI. Methods: We performed immunohistochemistry, real-time polymerase chain reaction analysis for mRNA and western blot analysis for protein to examine the time-dependent changes in SERT expression in the cerebrum during the first 14 days after TBI, using a controlled cortical impact model in rats. Results: SERT immunoreactivity in neuronal fibres within the area adjacent to the cortical contusion decreased 1 to 14 days after injury. Significantly decreased SERT mRNA and protein expression were noted in the area adjacent to the cortical contusion 7 days after injury. There were no significant changes in SERT expression in the cingulum of the injured brain. Discussion: The findings of this study indicate that TBI decreases SERT expression in the cerebral cortex. The decreased levels of SERT expression after TBI may result in decreased serotonin neurotransmission in the brain and indicate a possible relationship with depression following TBI.