Toshiro Kita

THE EXPRESSION OF TUMOR NECROSIS FACTOR-ALPHA IN THE RAT BRAIN AFTER FLUID PERCUSSIVE INJURY

Abstract To investigate the role of tumor necrosis factor-α (TNFα) after traumatic head injury in rats, moderate brain injury of 1000 mmHg was generated by an original fluid percussion injury device. TNFα levels in cerebrospinal fluid (CSF) gradually increased during the first 1 h, rose to a maximal elevation at 3 h and 6 h and returned to basal values by 24 h. Horseradish peroxidase tracer experiments revealed that primary microvascular damage appeared as early as 15 min after impact, but rapidly recovered and 1 h after impact secondary microvascular damage occurred in the hippocampus and parasagittal cortex. By immunoelectron microscopy, TNFα reactions were detected in the lysosomes of microglia accumulated at the impact site of the cortex 30 min after impact, and 1 h after impact these reactions were mainly detected at the glial cells (such as microglia and astrocytes) in the hippocampus and parasagittal cortex. Therefore the delayed microvascular damage observed in sites remote from the impact may be induced by TNFα which is synthesized mainly by glial cells. The present study suggests that TNFα conveyed from the microglial cells is one cofactor contributing to the fluid percussive brain edema formation after moderate brain injury.

Morphological study of fragmented DNA on touched objects

In recent years, forensic scientists showed that an individuals genetic profile can be retrieved from touched objects. Degraded DNA is believed to originate from epidermal cells and to be responsible for this phenomenon, yet the mechanism has not been confirmed. In the present study, we carried out a morphological and immunohistochemical investigation of nuclear DNA in differentiating keratinocytes in the skin and also a genetic analysis of DNA on swabs of human skin. Immunoelectron microscope analysis showed that single-stranded DNA was found both in the cornified layer of the skin and in swabs. Real-time-PCR assay proved that the DNA in the swabs was derived from the human DNA. Electron microscopic analysis of shadow-cast showed the presence of small DNA fragments in the swabs. It is conceivable that these DNA fragments on touched objects may originate from the epidermal cells of the cornified layer that are constantly sloughed off and leave for skin surface with sweat.

Role of p38 mitogen-activated protein kinase on cardiac dysfunction after hemorrhagic shock in rats.

Cardiac dysfunction is a well-known complication of hemorrhagic shock as a consequence of local inflammatory response. Several studies have indicated that p38 mitogen-activated protein kinase (MAPK) is a key mediator in organ dysfunction that is associated with the inflammatory state through the activation of proinflammatory cytokines such as TNF-&agr; and IL-1&bgr;. Whether the same applies to cardiac dysfunction after hemorrhagic shock has not been clearly determined. Therefore, in this study, the role of p38 MAPK on cardiac dysfunction after hemorrhagic shock was studied up to 5 h after a hemorrhage using FR167653, a specific inhibitor of p38 MAPK phosphorylation. The p38 MAPK phosphorylation, the cardiac mRNA expressions of TNF-&agr; and IL-1&bgr;, and intracardiac serum concentrations of each cytokine and creatine phosphokinase-MB isozyme increased after a hemorrhage. Activated neutrophil accumulation in the heart, histological inflammation-related injuries, and frequent ventricular arrhythmia were observed in the late phase after hemorrhagic shock. FR167653 inhibited these hemorrhagic changes except the induction of the primary hypotensive state. These results demonstrate that p38 MAPK phosphorylation in hemorrhagic shock plays an important role in the cardiac expression of the proinflammatory cytokines and in the development of cardiac dysfunction relative to the inflammatory responses.

The expression of tumour necrosis factor in the hypothalamus after treatment with lipopolysaccharide

To investigate the effects of tumour necrosis factor (TNF) in the hypothalamus, Wistar rats received an intravenous administration of lipopolysaccharide (LPS) at a dose of 3.0 mg/100 g. Concentrations of TNF‐α in the cerebral liquor and blood sera rapidly increased at 30 minutes after administration of LPS, rose to the maximum level at 1 hour, and then gradually decreased. Using horse‐radish peroxidase as a tracer, a transient increase in paracellular permeability throughout the tight junctions of the ependymal cell layer covering the third ventricle was observed by electron microscopy at 30 minutes and in that of the capillary endothelium at 1 hour after administration, respectively. Following LPS administration, TNF was preferentially localized by immunoelectron microscopy in the tight junctional area of the ependymal cell layer and the capillary. These data indicate that TNF, synthesized in the ependymal cell layer, induces a deterioration in the cerebrospinal fluid–brain barrier and subsequently in the blood–brain barrier. The present study suggests that oedematous changes in the hypothalamic areas determined by ultrastructural and magnetic resonance analyses were mainly due to TNF conveyed from the ependymal cell layer to the hypothalamus after administration of LPS.

The role of tumor necrosis factor-alpha in diffuse axonal injury following fluid-percussive brain injury in rats.

Abstract The immunolocalization of tumor necrosis factor-α (TNFα) after diffuse axonal injury (DAI) is demonstrated using a midline fluid percussion rat model (moderate brain injury of 1000 mm Hg was generated) and the effects of TNFα on the axolemmal permeability using horseradish peroxidase as a tracer. In addition, the accumulation of β-amyloid precursor protein (β-APP) was investigated, which has recently been shown to be a reliable marker for the diagnosis of DAI in cases with fatal head injury. TNFα levels in brain tissues from the impact site and the cortex including the corpus callosum, gradually increased during the first 1 h, rose to a maximal elevation at 3 h, gradually decreased at 6 h and decreased further at 24 h. Horseradish peroxidase (HRP) tracer experiments revealed that primary axonal damage appeared as early as 15 min after impact but rapidly recovered and that 1 h after impact, secondary axonal damage occurred in the corpus callosum and the brain stem. By immunoelectron microscopy it was seen that β-APP accumulated in the axon from 1 h after impact demonstrating that there was functional axonal damage. TNFα reactions were detected in the lysosomes of microglia 30 min after impact and 1 h after impact these reactions were mainly detected in the glial cells (such as microglia, astrocytes and oligodendrocytes) in the corpus callosum and the brain stem. It is generally accepted that TNFα directly induces primary demyelination and oligodendrocyte apoptosis. Therefore, TNFα conveyed from the microglial cells is one cofactor contributing to the formation of the delayed axonal damage observed at these sites. The present study suggests that TNFα conveyed from the glial cells may contribute to the pathogenic mechanism of DAI formation following fluid percussive brain injury.

Role of p38 mitogen-activated protein kinase pathway on renal failure in the infant rat after burn injury.

The p38 mitogen-activated protein kinase (MAPK) pathway is a proinflammatory signal transduction pathway for the production of cytokines and cellular response to stress, such as bacterial LPS or ischemia. We examined the effects of FR167653, a specific inhibitor of p38 MAPK, to explore the relationship between intestinal barrier damage and remote renal dysfunction. Immunohistochemical data showed the accumulation of neutrophils in the intestine after burn, and a horseradish peroxidase (HRP) tracer experiment showed burn-induced intestinal barrier damage. Our quantitative bacterial culture data demonstrated that viable bacteria reached the remote organs after burn and prevented the invading viable bacteria from using FR167653. Western blotting identified increased phosphorylation of p38 MAPK in the kidney after burn, and it may also have shown the possibility that endotoxin associated with the bacterial translocation enhances the activation of the p38 MAPK pathway. We blocked the intestinal barrier damage using FR167653, which resulted in reduced neutrophils in the intestine. FR167653 also prevented the increased phosphorylation of p38 MAPK in the kidney, which resulted in reduced neutrophils in the glomerulus and the reduction of tumor necrosis factor (TNF)-α and interleukin (IL)-1β mRNA in the kidneys, and, finally, prevented burn-induced renal failure. This study provides evidence for the hypothesis that the p38 MAPK pathway controls inflammatory mediators and not only improves intestinal function but also reduces remote renal failure after burn. We identified the pathophysiologic role of the p38 MAPK pathway in the development of renal failure after burn.

The immunocytochemical localization of tumour necrosis factor and leukotriene in the rat heart and lung during endotoxin shock

After the intravenous administration of lipopolysaccharide at a dose of 3.0 mg/100 g to rats, immunoreactive sites for tumour necrosis factor (TNF) and peptide leukotrienes (LTs) were examined in the heart and lung. Immunoreaction for TNF is preferentially localized on the apical endothelial cell surface of the vessels and in lysosomes of inflammatory and interstitial cells. Lysosomes of cardiac muscle cells which undergo degeneration are also reactive. Peptide LTs in inflammatory cells give almost the same reactions as those for TNF. However, the production of peptide LTs occurs uniquely in cardiac muscle cells in the media of the pulmonary vein, although lysosomes of intracardiac muscle cells which undergo degeneration do not show immunoreactivity. These results suggest that the degeneration of cardiac muscle cells may be induced not only by endogenous TNF but also by peptide LTs which are produced in muscle cells of the venous media and are transported to the myocardium via the coronary circulation.

Role of p38 mitogen-activated protein kinase pathway on heart failure in the infant rat after burn injury

We examined the hypothesis that post‐burn activation of the p38 mitogen‐activated protein kinase (MAPK) pathway is one aspect of the signalling cascade culminating in post‐burn secretion of tumour necrosis factor (TNF)‐α which contributes to post‐burn myocardial apoptosis. Studies were designed to determine the time course of the induction of p38MAPK, TNF‐α and myocardial apoptosis after burn injury. Our quantitative bacterial culture data demonstrated that viable bacteria reached the heart, and Western blotting data identified the increase in the phosphorylation of p38MAPK at an early time after burn. The peak incidence of myocardial apoptosis was also seen at an early time after burn. The expression of TNF‐α mRNA, infiltrated neutrophils and serum creatine phosphokinase myocardial band data peaked at a late time after burn. FR167653, a specific inhibitor of p38MAPK, prevented the induction of myocardial apoptosis, TNF‐α expression and myocardial injury after burn. Presumably, the bacterial LPS‐induced activation of p38MAPK pathway occurring at an early time after burn induced the subsequent myocardial apoptosis. The p38MAPK‐induced activation of pro‐inflammatory cytokine appeared to promote the degenerative myocardial injury at a late time after burn. Our present data provided evidence for the hypothesis that the p38MAPK pathway controls both myocardial apoptosis and the pro‐inflammatory mediator.

DISTRIBUTION OF PHENOL IN A FATAL POISONING CASE DETERMINED BY GAS CHROMATOGRAPHY/MASS SPECTROMETRY

A victim who was presumed to have ingested waste fluid containing phenol of DNA extraction was found dead in his laboratory. The skin was partially chemically burned, with blisters as maps. No mechanical injuries were observed. The pathological findings of the liver and kidney were typical of those of acute substantial poisoning. Phenol concentrations in the blood, urine, stomach contents and organs were determined by gas chromatography/mass spectrometry. Phenol was distributed throughout the body. The concentration of free phenol in the blood was found to be 60 micrograms/mL, and in the urine it was 208 micrograms/mL. The phenol concentrations in the organs were found as follows: 106 micrograms/g in the brain; 116 micrograms/g in the lungs; 166 micrograms/g in the liver, and 874 micrograms/g in the kidney, respectively. Significantly high concentrations were observed in the kidney, urine, and liver. To the best of our knowledge, such an intoxication through this kind of ingestion has never been reported before. Distributions of phenol in fatal poisonings have been reported, but colorimetry was used as the analytical method and it cannot exclude the interference of other phenolic compounds.

Sudden death due to acute pulmonary embolism from asymptomatic right atrial myxoma.

A 21-year-old man, in whom abnormal nodules had been detected in bilateral lung fields with no clinical symptoms for two years, was admitted to the hospital with sudden cardiopulmonary arrest. Acute pulmonary embolism with a large embolus was diagnosed, but the patient died soon after admission. When the examination was compared with X-ray taken 4 days earlier by chance, a large tumor was now detected in the right heart and was suspected to be the cause of this complication after his death. Necropsy found a 3.8 x 3.5 x 1.0 cm myxoid tumor arising in the right atrium and a large fragment of this type of tumor was at the pulmonary trunk. Many old myxoma fragments were noted in the bilateral peripheral branch of the pulmonary artery. It was concluded that the abnormal nodules were old pulmonary fragments and the cause of death was pulmonary embolism of a large fragment originated from the atrial myxoma. An asymptomatic right atrial myxoma is extremely rare but nevertheless possible to unexpected death like this case.