A quantitative study of lung dysfunction following haemorrhagic shock in rats.

Haemorrhagic shock occasionally causes an episode of lung dysfunction, the severity of which appears to correlate with fatal outcome. Our previous study indicated that proinflammatory cytokines, such as tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta, played a key role in the development of lung dysfunction through recruitment of activated neutrophils by causing pulmonary endothelial cell damage. Here, we examined this issue quantitatively by grading four groups of severity of bleeding in rats. As the amount of bleeding increased, the expression of mRNA for TNF-alpha and IL-1beta in the lung tissue and the pulmonary serum levels of both cytokines increased progressively up to 5 h, and the frequency of activated neutrophils increased likewise. The lung dysfunction indices serum lactic dehydrogenase-3 isozyme (LDH-3), partial pressure of arterial oxygen (PaO(2)) and alveolar-arterial oxygen tension difference (AaDO(2)) in blood deteriorated as the amount of bleeding increased. The frequency of activated neutrophils in the lung correlated well with the LDH-3 level 5 h after haemorrhagic shock. The present results demonstrate that the increase of proinflammatory cytokines and the recruitment of activated neutrophils into the lung following haemorrhagic shock are quantitatively related to progression of lung dysfunction as the amount of bleeding increases.

Perforation of the trachea by an endotracheal tube: an autopsy case.

An 80-year-old woman was intubated with a spiral endotracheal tube via a tracheal stoma during an arytenoidectomy. After being connected to the ventilator, the victim quickly became cyanosed, showed a decrease in blood pressure, and fell into cardiopulmonary arrest. Despite continuous resuscitation, the victim died. Necropsy found a tracheal perforation located 2.0 cm distal from the tracheal stoma that led to the right pleural space through the mediastinal space. We concluded that the tip of spiral endotracheal tube passed through the membranous part of the trachea into the pleural space and caused a hemopneumothorax followed by blood aspiration and death. Tracheal perforation is a rare, but life-threatening complication following a tracheostomal intubation. The inappropriate use of a tracheal tube stylet, guiding catheter, dilating forceps, and oversize tracheal tube have been demonstrated to cause airway injuries. However, fatal incorrect intubation with a spiral endotracheal tube via a tracheal stoma that resulted in death has not been reported previously.

[Autopsy case of drowning caused by accidental carbon dioxide intoxication in a hold tank].

A 49-year-old male captain fell and unfortunately died in a hold tank where he had entered to rescue his fainting co-worker on the disposing waste fluid left there. An autopsy revealed that the captain died from drowning in the waste fluid. In order to clarify the cause of their falling in the tank, the gas in the hold tank was analyzed. The concentration of oxygen was 18.86 to 19.31%, carbon dioxide was 7.28 to 9.07% and the other gases, including hydrogen sulfide, were assessed to be under the normal level. It was concluded that the intoxication of carbon dioxide generated from the waste fluid fermentation was the cause of this fatal accident through loss of consciousness. It is necessary to recognize that carbon dioxide is a dangerous and deleterious gas in circumstances where the gas can be produced.

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)-alpha which contributes to post-burn myocardial apoptosis. Studies were designed to determine the time course of the induction of p38MAPK, TNF-alpha 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-alpha 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-alpha 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.

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-alpha and IL-1beta. 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-alpha and IL-1beta, 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.

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

Hemorrhagic shock has been reported to induce renal dysfunction as a consequence of different kinds of local inflammatory response. p38 mitogen-activated protein kinase (MAPK) is a key mediator in organ dysfunction relating to the inflammatory states, and acts as an important mediator in the intracellular signal pathway for proliferation, differentiation, and production of proinflammatory cytokines such as TNF-alpha and IL-1beta. The effect of p38 MAPK on the hemorrhagic damage has not been clearly estimated as yet. In this study, our aim was to evaluate the role of p38 MAPK on the renal damage during the first 5 h after a hemorrhage using a specific inhibitor of p38 MAPK activation, FR167653. p38 MAPK activation increased immediately after a hemorrhage and decreased with time. renal mRNA expression of TNF-alpha and IL-1beta increased, renal dysfunction continued to progress, and histological inflammatory injuries were confirmed after hemorrhagic shock. With the pretreatment of FR167653, all of these hemorrhagic changes were attenuated, although the induction of the primary hypotensive state was confirmed. This study demonstrated that renal p38 MAPK is activated in hemorrhagic shock, promotes the expression of proinflammatory cytokines in the kidney, and consequently develops renal dysfunction. We concluded that p38 MAPK activation is essential in causing renal damage and that the inhibition of p38 MAPK activation blocks the development of the renal dysfunction after hemorrhagic shock.

Acephate in biological fluids of two autopsy cases after ingestion of the chemical.

Two autopsy cases, where the individuals were suspected of having ingested acephate, an organophosphorous insecticide, are reported. Acephate and its active metabolite, methamidophos (MP), were analyzed in the biological fluids by GC/MS, using the salting out method with liquid-liquid extraction columns. The first case was that of a 70-year-old man whose blood acephate was 149 microg/mL, and MP was 3.0 microg/mL. Serum pseudocholinesterase (ChE) activity was inhibited. No remarkable finding of injury or disease was determined as the cause of his death, but acute poisoning by acephate was mostly suspected. The second case was that of a 60-year-old man. A deep gash in the left neck injured the left common carotid artery in addition to the severely ischemic state of the primary organs. His blood acephate was 46 microg/mL, and MP was not detected. ChE activity was in the normal range. Hemorrhage was mainly suspected as the cause of his death. The concentrations of acephate and MP in human blood after oral ingestion are first reported here, and the acute toxic level of acephate is discussed.

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)-alpha and interleukin (IL)-1 beta 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.

Escherichia coli endotoxin enhances acute renal failure in rats after thermal injury.

This study was designed to evaluate the burned rat model to determine whether there are any differences in endotoxin-sensitive kidney functions between an infant rat (10-day-old pup) and an adult rat (10-week-old rat). Renal failure was observed in the infant burned rat and histological changes showed the adhesion of inflammatory cells in the glomerular capillaries and vacuolar changes in the renal proximal tubular cell. A horseradish peroxidase (HRP) tracer experiment suggested that the intestinal barrier damage of the infant burned rat was more severe than that of the adult burned rat. Therefore, more bacterial translocation of the intestinal flora, rich in endotoxin, might be expected in the infant versus the adult rats. Renal failure was not observed in the adult burned rat, so we investigated to determine the effects of endotoxin on the kidney function of the adult burned rat with low lethal lipopolysaccharide (LPS) or carrageenan (CAR). CAR is known to increase sensitivity to the lethal effects of endotoxin in rodents. Our present data demonstrated that renal failure was observed in the LPS- or CAR-treated adult burned rat and LPS- and CAR-treated adult rat (non-burned). These results show the possibility that endotoxin enhances renal failure in a burned rat model and provide additional support for the hypothesis that postburn renal failure is mediated, in part, by endotoxin associated with bacterial translocation.

Involvement of inducible nitric oxide synthase in renal failure after mild hemorrhage.

The role of iNOS in rats after mild hemorrhaging was examined in this study. A mild hemorrhage (17% of total blood) induced a decrease of systemic blood pressure and heart rate, transiently followed by gradual recovery. The hemorrhage caused expression of renal iNOS mRNA and an increase in systemic NO products at 1 h after bleeding. Serum creatinine and serum urea nitrogen (UN) increased progressively up to 5 h after bleeding. Light microscopic findings showed that some inflammatory monocytes, mainly consisting of neutrophil, often existed in the glomerular capillaries, eosinophilic changes were observed in the cytoplasm at the proximal tubules, and urinary casts existed in the uriniferous space at 5 h after bleeding. The selective iNOS inhibitor, S-methylisothiourea (MTU), suppressed hemorrhagic expression of renal iNOS mRNA and systemic NO products, suppressed the increases of serum creatinine and UN, and improved renal histological aggravations induced by hemorrhaging. We speculated that MTU caused the negative circuit to suppress the renal failure through a decrease of NO generation. These results in the present study showed that iNOS expression induced by mild hemorrhaging at the early phase did participate in the development of renal failure.

Marker of death from hemorrhagic shock.

Hemorrhagic shock has often been cited as a cause of death in forensic practical cases, but it is usually difficult to prove by autopsy findings alone. Our experimental studies demonstrated that hemorrhagic shock has promoted the endovascular recruitment of activated neutrophils and finally led to dysfunction of the shock organs. In this study, activated neutrophils were studied as a marker of death from hemorrhagic shock in the organs of autopsy cases in our department. Morphological changes in the heart, lung, liver, and kidney were examined in cases of death from hemorrhagic shock. The frequency of appearance of activated neutrophils was measured by immunohistochemical staining in the organs. The commonly observed morphological changes of hemorrhagic shock, which were not always expressed in any cases, could not account well for the cause of death. However, the expression of activated neutrophils in the organs elevated significantly at a middle and long antemortem interval. The appearance of activated neutrophils in the shock organs might be useful as a morphological marker of the cause of death from hemorrhagic shock in forensic practice cases.

Unexpected death in elephantiasis due to an abnormal life-style.

A 22-year-old man was found dead after he had continued to sit on a reclining chair for 2 years. He had consumed an unbalanced diet, kept wearing the same pair of socks and never washed himself for the term. His skin of bilateral crura developed into elephantiasis with severely festered ulcers on its surface. At autopsy, subcutaneous edema was significant in his lower limbs, and chronic circulatory disturbance of lymphoducts and veins was observed histologically. There were no crucial findings to account for chronic edema in the lower limbs. It has been reported that maintaining a seated posture obstructs both lymphoducts and veins because of bending the groin, decreases their return flow by inducing muscular atrophy, and causes subcutaneous edema in the lower limbs. Oligotrophia and dirt on his limbs might have exacerbated the chronic edema in elephantiasis. We concluded that a long-term abnormal life-style had caused fatal elephantiasis.

A study of neutrophil as a morphological marker of death from hemorrhagic shock in forensic practice cases.

Excessive autolytic inflammation accompanied by dysfunction of "shock organs" is recognized as arising from hemorrhagic shock due to the promotion of endovascular recruitment of neutrophils. Here, activated neutrophils in the organs of autopsy cases were evaluated as a marker of death from hemorrhagic shock. Morphologically-determined injury to the heart, lung, liver, and kidney was investigated in death from five major causes: hemorrhagic shock, head injury, exsanguination, asphyxia, and drowning. The frequency of activated neutrophils was assessed by immunohistochemical staining. When the antemortem interval was less than 2h, it was found that neither morphological damage nor neutrophil frequencies were significantly different after death due to any of these 5 causes. In contrast, at longer antemortem intervals up to 8h, the frequency of neutrophils in hemorrhagic shock was significantly greater than in head injury, whereas the degree of morphological damage was no different. Thus, the appearance of activated neutrophils in the primary organs could be useful to identify death caused by hemorrhagic shock after longer antemortem intervals.

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.

Role of tumor necrosis factor-alpha and interleukin-1beta on lung dysfunction following hemorrhagic shock in rats.

BACKGROUND: Hemorrhagic shock occasionally causes a fatal outcome following an outbreak of lung dysfunction, but the precise mechanism has not been clearly elucidated. Several studies have indicated that hemorrhagic shock causes a delayed vascular inflammatory decompensation and leads to inflammation-related organ dysfunction. Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta are known as major proinflammatory cytokines that play an important role in excessive autolytic inflammation, finally inducing organ dysfunctions. In this study, the role of TNF-alpha and IL-1beta on lung dysfunction following hemorrhagic shock was examined by using FR167653, a potent inhibitor of TNF-alpha and IL-1beta production that acts by suppressing p38 mitogen-activated protein kinase (MAPK). MATERIAL/METHODS: Hemorrhagic shock was induced in anesthetized male rats by bleeding via a common carotid catheter for 20 minutes to 25% of total body blood volume without fluid resuscitation. Mean blood pressure, heart rate and arterial blood gas components were recorded up to 5 hours after the bleeding. The levels of TNF-alpha, IL-1beta and lactic dehydrogenase (LDH)-3 isozyme were measured in the serum of pulmonary venous blood. The lung tissue was excised for the assay of mRNA and for histopathological study. RESULTS: The expressions of mRNA for TNF-alpha and IL-1beta in the lung tissue and the concentrations of both cytokines in pulmonary serum increased after a hemorrhage. Inflammation-related injuries and function deterioration were observed in the lung following hemorrhagic shock. These hemorrhagic changes were inhibited by pretreatment with FR167653. CONCLUSIONS: TNF-alpha and IL-1beta play a key role in the development of inflammation-related lung dysfunction following hemorrhagic shock. Our model should be useful to explain the pathogenesis of lung dysfunction following hemorrhagic shock.

Morphological study of fragmented DNA on touched objects.

In recent years, forensic scientists showed that an individual's 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.

The role of tumour necrosis factor-alpha in renal dysfunction following mild haemorrhage in rats.

Mild haemorrhage occasionally causes delayed death following failure of kidney or multiple organs, but the precise mechanisms have not yet been identified. We investigated the role of tumour necrosis factor-alpha (TNF-alpha), known as a major pro-inflammatory cytokine that leads to multiple organ failure, on the renal damage induced by mild haemorrhage. A mild haemorrhagic state was induced in male anaesthetized rats by bleeding via a common carotid catheter for 20 min at 16.7% of total body blood, 1.09 ml/100 g body weight, without fluid resuscitation. Mean arterial pressure and heart rate decreased soon after haemorrhaging but returned to baseline level up to 5 h after bleeding. TNF-alpha mRNA expression in the kidney and serum TNF-alpha levels were highest at 1 h after bleeding. Intraperitoneal pretreatment with FR167653, an inhibitory compound of TNF-alpha production, as well as of interleukin (IL)-1beta, significantly inhibited the increase in TNF-alpha. The inflammatory cell infiltration and tubular cell injury induced by haemorrhage were suppressed, and the renal dysfunction was dramatically improved by the FR167653 treatment. The morphological changes were also less in the treated group than in those that had not been treated. TNF-alpha has been reported to have striking effects on IL-1beta release and activation of neutrophils, and to play a pivotal role in the expression of the other pro-inflammatory cytokines. Our data show that endogenously-derived TNF-alpha does play a key role in the renal dysfunction during mild haemorrhage. These results should be useful to forensic pathologists to explain the pathogenesis of renal dysfunction induced by a mild haemorrhage and to identify the cause of death where there are no significant morphological changes after mild haemorrhage.