Ryouichi Tsuda

Induction of nitric oxide synthase by traumatic brain injury

We investigated the dynamic induction/expression of inducible nitric oxide synthase (iNOS) using human brains made available through death by traumatic brain injury (TBI). Astrocytes, microglia, and neutrophils were identified in tissue using immunohistochemical staining with antibodies against glial fibrillary acidic protein (GFAP), MHC class II antigen, and neutrophil elastase, respectively. The localization of iNOS protein in each of these cell types was evaluated using immunohistochemistry. Within 2 days of injury, iNOS immunoreactivity was not detected. However, after 2 days, immunoreactivity was detected in the traumatized brain. The iNOS immunoreactivity was localized on neutrophils and microglia/macrophages in the areas around the tissue necrosis in the traumatized cortical hemisphere, in the deep part of the cortex and the dentate gyri of the hippocampi adjacent to the hemorrhage, and within the cytoplasm of vascular smooth muscle cell of a small artery or arteriole surrounding the injured region. This reactivity was absent after 8 days post-injury.These observations confirmed the prolonged induction of iNOS within various cells in the injured brain. These responses suggest that iNOS plays a crucial role in cerebrovascular damage and/or secondary brain damage subsequent to traumatic brain injury. Furthermore, the dense nitric oxide (NO) generated by iNOS may play a role in neuronal cell death after injury.

Age-associated increases in heme oxygenase-1 and ferritin immunoreactivity in the autopsied brain

Heme oxygenase-1 (HO-1) is a 32 kDa heat shock protein (HSP) that catalyzes heme to biliverdin, free iron and carbon monoxide in the brain. Furthermore, the release of free ferrous ion by HO-1 plays an essential role in ferritin synthesis, and ferritin stores iron either for intracellular utilization, or for detoxification. It is well known that HO-1 immunoreactivity is enhanced greatly in neurons and glia of the hippocampus and cerebral cortex in various pathophysiological conditions. The expression of HSP 70 is well known for the age-associated increase, but the expression modalities of HO-1 and ferritin associated with aging are still unknown. A study was therefore performed to examine the correlations in the expression of HO-1 and ferritin with age using immunohistochemistry. We investigated 31 autopsied brains (3-84-year-olds) without traumatic brain injury and neurodegenerative disease. The specimens were taken from the cerebral cortex and hippocampus. In the cerebral cortex, age (aging) had a statistically significant positive correlation with HO-1 (r=0.894, P<0.01) and ferritin (r=0.731, P<0.01). In the hippocampus, age had a significant positive correlation with only HO-1 (r=0.660, P<0.01). These results showed that HO-1 and ferritin underwent an age-related increase in human brain, especially in the cerebral cortex. Our results also indicate that various stress responses may persist in the aged human brain.

The time-course analysis of gene expression during wound healing in mouse skin.

RNA analysis has been applied to forensic work to determine wound age. We investigated mRNA expression using quantitative RT-PCR of ten genes, including c-fos, fosB, mitogen-activated protein kinase phosphatase-1 (MKP-1), CD14, chemokine (C-C motif) ligand 9 (CCL9), placenta growth factor (PlGF), mast cell protease-5 (MCP-5), growth arrest specific 5 (Gas5), beta-2 microglobulin (B2M) and major urinary protein-1 (MUP-1), in terms of repair response in adult mice. The expression level of c-fos, fosB and MKP-1 transcripts increased drastically, peaked within 1h, and that of the CD14 and CCL9 transcripts peaked from 12 to 24h. An increase in PlGF and MCP-5 mRNA appeared on about day 5. Gas5, B2M and MUP-1 transcripts showed no significant change. Each gene had differentially expressional patterns with time-course. Our result implied that the observation of the 7 genes in wounded skin could serve to aid in the accurate diagnosis of wound age.

The expression of excitatory amino acid transporter 2 in traumatic brain injury

It is well recognized that glutamate is the major excitatory neurotransmitter, which is removed from the synaptic cleft by excitatory amino acid transporter 2 (EAAT2) located on the perisynaptic astrocytes and that neuronal death has been associated with an increased extracellular glutamate concentration. In this study, we have immunohistochemically demonstrated the expression of EAAT2 protein in the human brain after traumatic brain injury (TBI). The EAAT2 expression patterns can be divided into three types: continuous and highly extensive staining (E); continuous but sporadic staining (M); and sporadic pattern staining (S). In six of the nine short survival cases studied (1 h to 1 day), continuous and highly extensive staining for EAAT2 (E type) was observed in the ipsilateral cerebral cortex. On the other hand, we were able to demonstrate weak staining (S and M types) in 5 of the 7 long survival cases (> or =1 day) and in 12 of the 14 very short survival cases (<1 h) studied. Similar findings were obtained in the contralateral cerebral cortex and also in the ipsilateral hippocampus. In addition, positive staining for glial fibrillary acidic protein was detected around the cerebral contusion, but the EAAT2-positive expression was not observed in the same region for all of the six short and long survival cases (> or =1 h) after TBI. These findings clearly showed the differences in EAAT2 expression in the cerebral cortex according to the survival time and severity of cerebral contusion after TBI. Therefore, we emphasized that EAAT2 might play an important role in contributing to extracellular glutamate concentrations and secondary brain injury after TBI.

The expression of excitatory amino acid transporter 2 (EAAT2) in forensic autopsy cases

Glutamate is the major excitatory neurotransmitter and the greater part of this amino acid is removed from the synaptic cleft by excitatory amino acid transporter 2 (EAAT2) located on perisynaptic astrocytes. Recently, it was reported that the EAAT2 protein content changed in rats following forebrain ischemia and administration of methamphetamine. We planned to demonstrate the immunohistochemical distribution of EAAT2 in the human brain and discuss the significance of its pathophysiological roles. Thirty-two cases were used from forensic autopsies. The tissues were sampled from the cerebral cortex, striatum and hippocampus. The distribution of EAAT2 was difficult to identify in cases of electrical fatalities. However, continuous and extensive staining of EAAT2 was observed in cases of death from hypothermia. In almost all asphyxia death, we were able to observe a weak stain of EAAT2. In case of solvent abuse, EAAT2 staining was continuous and extensive as in the cases of hypothermia, and patchy negative zones were mixed. This study clearly showed the differences in EAAT2 localization according to the cause of death. These findings suggested that the differences in EAAT2 staining depended on the cause and course (pathophysiological conditions) of death.

Immunohistochemical study on the induction of heme oxygenase-1 by traumatic brain injury.

We investigated the dynamics of the induction of heme oxygenase-1 (HO-1) in the human brain after death caused by traumatic brain injury (TBI). HO-1 was found to stain neurons and microglia/macrophages in cases with TBI, whereas no positive staining except for a few round cells in the arachnoidal space was observed in control cases. In a case with 7h survival, a considerable number of HO-1 positive neurons and microglia were observed. The number of HO-1 positive cells and level of HO-1 staining gradually increased up to 24h survival. Although HO-1 positive neurons were seldom observed in cases with more than 7-day survival, HO-1 positive microglia were still observed even in cases with 5-month survival. The results indicate that HO-1 may be induced by TBI in human cases, and suggest that prolonged HO-1 induction in microglia might reflect its role in protecting those cells from secondary damage including oxidative stress.

The expression of a novel stress protein ‘150-kDa oxygen regulated protein’ in sudden infant death

The oxygen regulated protein 150-kDa (ORP-150) is only induced in hypoxic conditions. We performed an immunohistochemical and morphometrical study on the expression of ORP-150 in the brains of sudden infant death (SID) victims. The cerebral cortexes of 18 infants were used for this study. Each tissue section was incubated with anti-ORP-150 polyclonal antibodies and the number of ORP-150 positive cells was counted. In the cluster analysis, the 18 cases were classified into three groups (A-C groups). Group A was composed of six sudden infant death syndrome (SIDS) cases and its mean value of ORP-150 positive cells was 66.75+/-3.44, Group B (six severe respiratory infectious disease such as pneumonia and bronchitis including sepsis): 39.50+/-2.52 and Group C (five SIDS and one severe respiratory infectious disease): 16.00+/-2.92, respectively. These results might reflect chronic hypoxic condition before death, because ORP-150 is only induced when a hypoxic condition exist, but not acute hypoxia. And chronic hypoxic state is likely to be antecedent to SIDS. Therefore, immunohistochemical analysis of OPR-150 in the brain of SID cases may be very useful to differentiate between SIDS and acute asphyxia.

Increase in Dual specificity phosphatase 1, TGF-beta stimulated gene 22, domain family protein 3 and Luc7 homolog (S. cerevisiae)-like messenger RNA after mechanical asphyxiation in the mouse lung

We investigated the transcriptome profile of mechanical asphyxia and decapitation at 60 min after death using serial analysis of gene expression. After comparing the results, 11 genes were significantly increased by the mechanical asphyxia treatment in the mouse lung. Of those genes, quantitative real-time PCR revealed that dual specificity phosphatase 1 (Dusp1), TGF-beta stimulated gene 22, domain family protein 3 (TSC22d3) and Luc7 homolog (Saccharomyces cerevisiae)-like (Luc7l) after asphyxia were more significantly increased than those after decapitation. Dusp1 inactivated mitogen activated protein kinase, which functions in cell proliferation. However, the consumption of oxygen had a disadvantageous effect on survival, because tissue or cells were not able to produce energy by internal respiration under the suddenly hypoxic condition following asphyxia. The increased transcripts of Dusp1 following asphyxia suppressed oxygen consumption. TSC22d3 was isolated as a TGF-beta-inducible gene and it is also identified as a glucocorticoid (GC)-induced leucine zipper (GILZ). GC was released from the adrenal gland via HPA axis under the hypoxic condition. Especially in acute suffocation, GC rapidly increased. Therefore, the increase in TSC22d3 may be induced by the increased GC following asphyxia. We were unable to clarify the Luc7l increase, because there are no reports in relation to asphyxia. In addition, GILZ mediates the antiproliferative activity of glucocorticoids. We thought that the increasing TSC22d3 may lead to the suppression of oxygen consumption to avoid wasting energy, as in proliferation, the same as the increase in Dusp1. Our data indicated that the determination of the protein product level in the lung could help in diagnosing asphyxia. In addition, these data may contribute to revealing the patho-physiology of asphyxia and to help diagnose asphyxia, including hanging.

Hereditary recombined three-allele variant of the Gc system

A three-allele variant with Gc 2, Gc 1F and Gc 1A2 alleles was detected in both a baby and his mother during paternity testing by isoelectric focusing. His father had a normal Gc phenotype, Gc 2-1F. Further examination of his mothers relatives revealed that his grandfather also had the same three-allele variant, while his grandmother and his aunt had normal Gc 2-1F and Gc 2-2. From these results, it was considered that the Gc 1F and Gc 1A2 alleles were on the same single chromosome. It was suggested that recombination had occurred between two chromosomes that had the Gc 1F and Gc 1A2 allele, respectively, forming the variant allele Gc 1F1A2 on a single chromosome.

Immunohistological investigations of autopsied carotid bodies and their application to diagnosing strangulation

SummaryUsing immunohistochemical staining, the histological changes and the presence of neuropeptides (enkephalin and VIP) in the carotid body have been investigated in medico-legal autopsy cases, especially asphyxia cases. Only in cases of manual and/or ligature strangulation cases that sustained a force near the carotid body, were the chief cells mainly lightly stained, indicating that they had been “active” cells. Furthermore, these cells and their nuclei were enlarged in comparison to the chief carotid body cells in other autopsy groups. It was thus felt that these changes had resulted from the force that had directly affected the carotid body. Based on these findings, it was concluded that immunohistochemical investigation of the carotid body offers a useful possibility for diagnosing manual asphyxia, especially in autopsy cases involving strangulation.ZusammenfassungMit Hilfe immunhistochemischer Färbemethoden wurden die histologischen Veränderungen und das Vorhandensein von Neuropeptiden (Enkephalin und VIP) im Glomus caroticum bei rechtsmedizinischen Autopsie-Fällen, speziell bei Asphyxie-Fällen, untersucht. Lediglich in Fällen von manueller und werkzeugbedingter Strangulation, in denen die Gewalt in der Nähe des Glomus caroticum erlitten wurde, waren die Hauptzellen hauptsächlich leicht angefärbt, als Hinweis, daß sie „aktive” Zellen darstellten. Weiterhin waren diese Zellen und ihre Kerne vergrößert im Vergleich zu den Hauptzellen des Glomus caroticum in anderen Autopsie-Fällen. Es entstand daher der Eindruck, daß diese Veränderungen resultierten aus der Gewalt, die direkt das Glomus caroticum traf. Aufgrund dieser Befunde wurde geschlossen, daß die immunhistochemische Untersuchung des Glomus caroticum eine nützliche Möglichkeit darstellt, um die manuelle Asphyxie zu diagnostizieren, speziell in Autopsie-Fällen unter Einbeziehung der Strangulation.