Ichiro Nakasono

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.

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.

An asthmatic death while under Chinese acupuncture and moxibustion treatment.

A 29-year-old Japanese man with bronchial asthma died while undergoing Chinese acupuncture and moxibustion treatment. The autopsy findings of the lungs were compatible with a diagnosis of severe asthma. Further, on immunohistochemical examination, hypoxic brain damage and an unusual distribution of pulmonary surfactant were found. In contrast, only minor hemorrhages in the right semispinal muscle and round-shaped bruises were seen as a result of Chinese acupuncture and moxibustion treatment. Thus, it was concluded that the man had died from a severe asthmatic attack

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.

C-fos, fos-B, c-jun and dusp-1 expression in the mouse heart after single and repeated methamphetamine administration

In forensic autopsy, there are numerous sudden methamphetamine (MA)-related deaths. The concentration of MA in the blood is measured to determine the cause of death in case of MA-related death. As a low concentration of MA is detected in MA-related death cases, it is sometimes difficult to identify to the cause of death. MA abusers often exhibit various cardiovascular diseases. MA induces arrhythmia and morphological change in cultured cardiomyocytes. Therefore, MA might affect heart cells, especially in terms of gene expression. Immediate early genes (IEGs) are expressed before some specific gene expressions following certain stimuli. We investigated the expression of IEGs, including c-fos, fos-B, c-jun and dusp-1 mRNA, in the mouse heart after a once-daily MA injection for 1day, 2 or 4weeks using real-time quantitative PCR. We showed that high-dose (10mg/kg) MA administration on day 1 induced mRNA expression of the four IEGs. In contrast, low-dose (1mg/kg) administration on day 1 did not induce any c-fos expression. These findings were characteristic only of the heart, since c-fos increased after treatment at any dose in the brain, suggesting that the intracellular signal cascade differs in these two organs. Nevertheless, we confirmed the transcriptional tolerance in the heart as well as the brain on chronic administration by investigating IEG expression. We were unable to explain why the expressions of IEGs were similar between both doses of MA after chronic administration, although these differed after the single treatment. Additionally, these results strongly suggest that the transcriptome must be altered after long-term treatment. As MA abuse results in various cardiovascular diseases, investigation of the transcriptome in the heart after chronic MA administration will aid in elucidating the patho-physiology of MA-related cardiovascular disease.

Induction of apolipoprotein E after traumatic brain injury in forensic autopsy cases.

We investigated the dynamics of the induction of apolipoprotein E (apoE) in the human brain after death caused by traumatic brain injury (TBI). A striking difference in apoE immunoreactivity in the traumatised cortical hemisphere compared with the contralateral non-traumatised hemisphere was observed. ApoE was detected within the neurons of the traumatised cortical hemisphere in cases surviving only about 2 h, as well as in those surviving for extended periods. In contrast, no apoE staining within the neurons was seen in the contralateral cortical hemisphere. ApoE staining within astrocytes was faint in both traumatised and contralateral hemispheres of cases surviving only 2 h. However, staining was intense in the traumatised hemispheres in short as well as long surviving cases, even those surviving more than 1 month. ApoE immunoreactivity was also observed in areas adjacent to capillaries and surrounding the neuropil of the injured hemisphere. These observations corroborated the idea of a prolonged induction of apoE within the neurons and also in the extracellular matrix after TBI. Furthermore, the possibility is suggested that the alteration of apoE distribution may contribute to a cerebroprotective mechanism immediately after TBI.

An autopsy case of neuroleptic malignant syndrome (NMS) and its immunohistochemical findings of muscle-associated proteins and mitochondria

Neuroleptic malignant syndrome (NMS) is a rare but potentially fatal disorder. In forensic cases, post-mortem diagnosis of NMS is sometimes difficult if ante-mortem information, such as neuroleptic ingestion or signs and symptoms, cannot be obtained. A 39-year-old Japanese male on a neuroleptic treatment regimen suddenly became agitated and died. Autopsy revealed muscle rigidity and hyperthermia. Post-mortem examination of blood revealed elevation of creatine phosphokinase-MM (CK-MM) and lactate dehydrogenase-4 and dehydrogenase-5 (LDH-4 and LDH-5). In renal glomeruli and tubules, myoglobin was stained immunohistochemically. From these findings, the cause of death was considered to be NMS. To support the diagnosis of NMS, both skeletal and cardiac muscles were stained with actin, myoglobin, desmin and mitochondria antibodies immunohistochemically. Actin, myoglobin, desmin, and mitochondria had been lost from skeletal, but not from the cardiac muscle, which suggested that only the skeletal muscle was damaged. Moreover, because mitochondria had disappeared only from the skeletal muscle, it was considered that skeletal muscle degeneration was caused by mitochondrial damage. Therefore, it is suggested that immunostaining of skeletal muscle by antibodies for muscle-associated proteins and mitochondria is useful to corroborate a diagnosis of NMS.

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.

A demonstration of spermatozoa on vaginal swabs after complete destruction of the vaginal cell deposits.

The proteolytic enzyme, proteinase K, has been found to destroy all vaginal cells though it does not have the same effect on spermatozoa. In cases of sexual offenses, in which a swab has been used to wipe out the vagina, the female cells and their nuclei on that swab may also contain the heads of spermatozoa. After as short a time as 30 min of proteinase K treatment, the spermatozoa that had separated from the enzymatically destroyed vaginal cells were recovered. This proteinase destruction furnishes some spermatozoa with deformed heads and a somewhat greater number of isolated tails though a sufficient number of spermatozoan heads still remain for a reliable diagnosis. For detection of spermatozoa from a vaginal swab after proteinase K pretreatment, the heads of the spermatozoa are distinctly stained by Oppitzs method. Further, on prior treatment with proteinase K, the ABO blood grouping of the spermatozoa could also be determined on the vaginal swab by using the absorption-elution technique. The resistance of the spermatozoa to proteinase K is the basis for this method.

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.