Dimethylthiourea ameliorates carbon tetrachloride-induced acute liver injury in ovariectomized mice.

AIMS: In order to clarify hepato-protective actions of estrogen, we examined the progress of carbon tetrachloride (CCl4)-induced acute liver injury (ALI) in sham and ovariectomized (ovx) mice and the effects of dimethylthiourea (DMTU), a hydroxyl radical scavenger, and meloxicam (Melo), a selective cox-2 inhibitor, on the development of CCl4-induced ALI. MAIN METHODS: Female C57BL/6 J mice weighing 15-20 g were performed sham or ovx operation at 8 weeks of age. Blood and liver samples were collected 15 and 24 h after CCl4 administration. Sham and ovx mice were given DMTU, Melo or saline intraperitoneally 30 min before CCl4 or corn oil administration. KEY FINDINGS: ALT levels in ovx mice were significantly increased compared to those in sham mice. DMTU reduced ALT levels in ovx mice to the same levels as those in sham mice after CCl4 injection. CCl4 upregulated TNF-α, IL-6, cox-2 and iNOS expression in ovx mice compared to the levels in sham mice. DMTU significantly reduced cox-2 and iNOS expression levels upregulated by CCl4 in ovx mice. However, pretreatment with Melo had no effects on ALT levels and the gene expression levels of TNF-α, IL-6 and HO-1 in either sham or ovx mice, indicating that cox-2 may not participate in increase of CCl4-induced ALI caused by estrogen deficiency. SIGNIFICANCE: Ovariectomy accelerated the development of CCl4-induced acute liver injury, and DMTU reduced liver injury. These results suggest that estrogen may act as an antioxidant in the development CCl4-induced acute liver injury.

Apocynin reduced doxycycline-induced acute liver injury in ovariectomized mice.

To determine the physiological role of estrogen in the development of liver injury, we examined the sensitivities of sham and ovariectomy (ovx) mice against doxycycline (DOXY)-induced acute liver injury. Ovx or sham operation was performed in C57BL/6J wild-type female mice of eight weeks of age. Sham mice and ovx mice were treated with DOXY (240 mg/kg ip) 8 weeks after the operation, 30 min after apocynin (5 mg/kg) or saline administration. Blood and liver samples were obtained at 3 and 6 h after DOXY administration. Liver dysfunction occurred soon after DOXY administration and became more severe in ovx mice than in sham mice. At early phase after DOXY injection, TNF-α and iNOS inductions upregulated almost the same levels in sham and ovx mice. On the other hand, expression levels of IL-6, IL-10, c-fos, cox-2 and HO-1, downstream genes of TNF-α, were significantly increased in ovx mice compared to those in sham mice, correlated with liver dysfunction. In addition, apocynin, a NADPH oxidase (Nox) inhibitor, totally improved DOXY-induced liver injury in both sham and ovx mice, indicating that reactive oxygen species generated through Nox activation by DOXY are responsible for development of acute liver injury.

Oxidative stress may be involved in distant organ failure in tourniquet shock model mice.

Crush syndrome is characterized by prolonged shock resulting from extensive muscle damage and multiple organ failure. However, the pathogenesis of multiple organ failure has not yet been completely elucidated. Therefore, we investigated the molecular biological and histopathological aspects of distant organ injury in crush syndrome by using tourniquet shock model mice. DNA microarray analysis of the soleus muscle showed an increase in the mRNA levels of Cox-2, Hsp70, c-fos, and IL-6, at 3h after ischemia/reperfusion injury at the lower extremity. In vivo staining with hematoxylin and eosin (HE) showed edema and degeneration in the soleus muscle, but no change in the distant organs. Immunohistological staining of the HSP70 protein revealed nuclear translocation in the soleus muscle, kidney, liver, and lung. The c-fos mRNA levels were elevated in the soleus muscle, kidney, and liver, displaying nuclear translocation of c-FOS protein. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) analysis suggested the involvement of apoptosis in ischemia/reperfusion injury in the soleus muscle. Apoptotic cells were not found in greater quantities in the kidney. Oxidative stress, as determined using a free radical elective evaluator (d-ROM test), markedly increased after ischemia/reperfusion injury. Therefore, examination of immunohistological changes and determination of oxidative stress are proposed to be useful in evaluating the extent of tourniquet shock, even before changes are observed by HE staining.

Interleukin-6 modulates oxidative stress produced during the development of cisplatin nephrotoxicity.

AIMS: We reported that interleukin-6 (IL-6) plays a protective role in the development of cisplatin-induced acute renal failure (ARF) through upregulation of anti-oxidative stress factors. In this study, we examined the effects of dimethylthiourea (DMTU), a hydroxyl radical scavenger, on the development of cisplatin-induced ARF in wild-type (WT) and IL-6(-/-) mice to determine how IL-6 contributes to modulation of oxidative stress caused by cisplatin. MAIN METHODS: WT and IL-6(-/-) male mice were given either cisplatin (30 mg/kg) or saline intraperitoneally. DMTU (100mg/kg) or saline was given 30 min before cisplatin or saline administration. Blood and kidney samples were collected on days 1 and 3 after cisplatin administration. KEY FINDINGS: In WT mice, DMTU markedly improved cisplatin-induced renal dysfunction and survival rate. DMTU reduced the expression levels of TNF-α, Bax and c-fos and increased the expression levels of IL-6, Bcl-xL and Nrf2 in WT mice. Reduced reactive oxygen species (ROS) by DMTU resulted in increases of IL-6, anti-apoptosis and anti-oxidant gene expression levels. In IL-6(-/-) mice, DMTU also improved cisplatin-induced renal dysfunction and reduced expression levels of TNF-α, Bax and c-fos, but not Bcl-xL and Nrf2. Since Nrf2 induces IL-6 expression, IL-6 and Nrf2 may influence each other during anti-oxidant responses. The basal level of HO-1 in IL-6(-/-) mice was higher than that in WT mice. SIGNIFICANCE: In IL-6(-/-) mice, overproduction of ROS by cisplatin results in upregulation of HO-1 expression in order to eliminate oxidative stress. IL-6 mediates the generation and elimination of ROS during cisplatin-induced ARF.

Interleukin-6 plays a protective role in development of cisplatin-induced acute renal failure through upregulation of anti-oxidative stress factors.

AIMS: Cisplatin, a major chemotherapeutic agent, accumulates in proximal tubules of the kidneys and causes acute renal failure dose-dependently. We previously reported that cisplatin induced more severe renal dysfunction in interleukin-6 (IL-6) knockout (IL-6(-/-)) mice than in wild-type (WT) mice. Expression of a pro-apoptotic protein was significantly increased with cisplatin in IL-6(-/-) mice compared to that in WT mice. IL-6, locally expressed in renal tubular cells after cisplatin administration, prevents the development of renal dysfunction at an early stage. In the present study, we focused on downstream signals of IL-6 and oxidative stress induced by cisplatin in order to evaluate the protective role of IL-6 in the development of acute renal failure. MAIN METHODS: WT and IL-6(-/-) mice were given either cisplatin (30 mg/kg) or saline intraperitoneally. Blood and kidney samples were collected at 24h and 72 h after cisplatin administration. The changes in expression of 4-hydroxy-2-nonenal protein (4-HNE, oxidative stress marker) and cyclooxygenase-2 (cox-2), activities of superoxide dismutases and caspase-3, and phosphorylation of extracellular signal-regulated kinase (ERK) were examined. KEY FINDINGS: Cisplatin increased the expression of 4-HNE and cox-2, and phosphorylation of ERK in IL-6(-/-) mice than in WT mice. On the other hand, activity of superoxide dismutase, an anti-oxidative enzyme, was significantly decreased in the kidney obtained from IL-6(-/-) mice after cisplatin administration. SIGNIFICANCE: Our findings suggest that IL-6 plays a protective role in the development of cisplatin-induced acute renal failure through upregulation of anti-oxidative stress factors.

Fatal human poisoning with Padan: a cartap-containing pesticide.

We report a patient who ingested about 13 g of Padan SG, a cartap-containing pesticide. After ingestion, the patient developed multiple seizures and dyspnea and lost consciousness. The patient did not recover and died on the fifth hospital day despite treatment at the early stage of poisoning. The cause of death was multisystem organ failure. Results of toxicological analysis were as follows: concentrations of nereistoxin (cartap metabolite) were 10.6 microg/mL in plasma, 18.2 microg/mL in urine, and 2.6 mg/mL in gastric fluid. Results of drug screening of urine by Triage DOA Panels and using an organophosphate detection kit were negative.

Comparison of renal dysfunction in wild-type, IL-6 KO and iNOS KO mice hind limb tourniquet-reperfusion model.

The release of a tourniquet after hind limb ischemia results in vital organ injury, which progresses to multiple organ failure with a high mortality rate. Many events are involved in ischemia-reperfusion (I/R) injury. The purpose of this study was to determine how IL-6 or iNOS is involved in I/R injury using IL-6 knockout (KO) and iNOS KO mice. Male C57BL/6J wild-type (WT), IL-6 knockout (KO) and iNOS KO mice were anesthetized with pentobarbital, and rubber bands were fastened to the inguinal region of both hind limbs for 3h. Blood and kidney samples were obtained before reperfusion and at 1, 2, 3, and 12h after reperfusion. For the control group, mice were kept for 6h under an anesthetized condition without rubber bands. Blood gases and biochemical parameters were analyzed by i-STAT300F. Real-time PCR analyses were performed to examine the expression levels of IL-6 and iNOS mRNA in kidneys. Metabolic acidosis, hemoconcentration and renal dysfunction were significantly developed after reperfusion regardless of mouse genotype, and progression of this condition was earlier in IL-6 KO and iNOS KO mice than in WT mice. The expression level of kidney IL-6 mRNA increased and that of iNOS mRNA decreased after reperfusion. It is possible that late decrease recovery of iNOS mRNA expression in IL-6 KO mice and early progress of IL-6 mRNA expression in iNOS KO mice after reperfusion induce renal dysfunction.

PCR detection of bacterial genes provides evidence of death by drowning.

We have developed a sensitive and specific PCR method for detecting plankton DNA in cases of death by drowning. However, this PCR method could not be used for cases of drowning in water containing no plankton. Bacteria species are normally localized in the throat and trachea and they may invade into blood through the respiratory tract in people who have drowned as well as species localized in water. The aim of this study was to establish a novel and expedient PCR method for detecting bacterial genes in samples from drowning cases. We designed primer pairs for Streptococcus salivarius (SL1) and Streptococcus sanguinis (SN1), which are common species in the throat, and for Aeromonas hydrophila (AH1), which has been found in various water samples. With SL1, SN1, and AH1, we detected 10, 0.1, and 1 pg of target DNA, respectively. Among 19 drowned cases within 3 days postmortem, SL-DNA was detected in all of the blood samples from hearts with SL1 and AH-DNA was detected in several samples with AH1. In a case of drowning in a bathtub, use of the conventional acid digestion method for diatom analyses and the PCR method for identifying plankton DNA revealed no plankton, but our PCR method for detecting bacterial DNA showed a positive result for SL-DNA in a blood sample from the heart. In conclusion, our novel PCR method is highly specific and sensitive for detecting bacterial DNA and is useful for cases of death by drowning in water containing no plankton.

Western blot analysis for 4-hydroxy-2-nonenal (HNE)-modified proteins in paraquat-treated mice.

Paraquat (PQ) is widely used in agriculture as a non-selective contact herbicide. Ingestion of PQ results in multiple organ failure within one week, although the primary damage induced by PQ occurs in the lungs. It is known that reactive oxygen species (ROS) play an important role in pathological changes in PQ poisoning, although the exact mechanism of PQ toxicity has not been completely elucidated. In this study, we investigated changes in 4-hydroxy-2-nonenal (HNE)-modified proteins as markers of lipid peroxidation in PQ-treated mice. C57BL/6J mice were given PQ (10 or 50 mg/kg) intraperitoneally. After 24 h, blood and tissues were collected under isofluorene anesthesia. For histochemical studies, frozen tissue sections were immunostained with an anti-HNE monoclonal antibody. Immunoreactivity using the anti-HNE antibody was strongly increased in the kidney after PQ treatment. The expression levels of HNE-modified proteins in tissue homogenates were analyzed by Western blotting. Tissue homogenates were separated by 12.5% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. The membrane was immunostained with an anti-HNE antibody. Reactive bands were visualized with diaminobenzidine (DAB). Then the membrane was immunostained again with an anti-actin antibody and visualized with New Fuchsin. An anti-actin antibody-positive band was used to correct the protein content in tissue homogenates. The intensity of the 41-kDa protein band in the kidney and that of the 51-kDa protein band in the lung were significantly increased. These findings suggest an important role of ROS in the development of paraquat toxicity in the kidney and lung. On the other hand, an increase in peroxidation was not observed in the liver protein. It has been reported that oxidative stress after paraquat administration involved nitration of proteins by the activation of nitric oxide synthase (NOS). Therefore, paraquat may exert its toxicological action on different organs by different mechanisms.

Histologic study of arteriovenous shunts in the normal dura mater adjacent to the transverse sinus.

BACKGROUND: Although AV shunts are known to exist in the normal dura mater, their histologic appearance has not been examined in detail. Arteriovenous shunts in the normal dura mater adjacent to the transverse sinus, the most frequent site of DAVFs, were studied histologically. METHODS: Normal dura mater adjacent to the transverse sinus was obtained from 8 cadavers. Each specimen was cut into approximately 4-microm-thick serial sections; these were stained by the elastic Masson method and examined under a light microscope. RESULTS: Of the 8 specimens, 5 harbored a total of 6 AV shunts; no shunts were found in the other 3 specimens. The shunts, located in the supratentorial (n = 1) and infratentorial dura mater (n = 4) and in the tentorium cerebelli (n = 1), were classified into 2 types. In direct-type shunts, the artery connected directly to the vein; the diameter of these shunts ranged from 40 to 80 microm. In indirect-type shunts, the artery and vein were parallel and were indirectly connected by a shunt vessel, producing an H shape. The diameter of these shunt vessels ranged from 30 to 45 microm. All 6 shunts were connected to veins or the venous lake; none connected directly to the transverse sinus. CONCLUSIONS: The existence of direct- and indirect-type AV shunts in the normal dura mater was confirmed histologically. Both types exhibited the histologic features of DAVFs, suggesting that AV shunts in the normal dura mater might be involved in the etiology of DAVFs.

Enhanced lipid peroxidation in tourniquet-release mice.

BACKGROUND: The pathogenesis of ischemia-reperfusion involves generation of reactive oxygen and resulting lipid peroxidation. However, investigation that ischemia-reperfusion following tourniquet release enhances lipid peroxidation is insufficient. METHODS: Tourniquet was applied to a unilateral hind limb of mice for 3h followed by 5-, 15-, 30- and 60-min release. To examine superoxide production immunohistochemically in ischemia-reperfusion muscles, a primary antibody directed to 4-hydroxy-nonenal (HNE) was used. Furthermore, we analyzed 7alpha- and 7beta-hydroperoxycholest-5-en-3beta-ol, 7alpha- and 7beta-hydroxycholesterol, and 7-ketocholesterol by HPLC in the gastrocnemius muscles, kidneys, liver, heart and lungs of mice after 1-h reperfusion. RESULTS: Increased HNE immunoreactivitiy was observed in the tourniquet-applied side of gastrocnemius muscles of hind limb particularly after 5-min reperfusion. All the oxysterols were significantly higher in the gastrocnemius muscles of the tourniquet-applied side than of the contralateral muscles. Oxysterols were elevated in the kidneys and the liver. Together with the presence of high blood urea nitrogen, these data indicate that the kidney is vulnerable to ischemia-reperfusion. CONCLUSIONS: The enhanced oxidative stress due to ischemia-reperfusion appears to increase HNE in muscle and oxysterols by peroxidation not only in the gastrocnemius muscles but also in the kidneys and liver.

A hind limb tourniquet induces interleukin-6 expression in a rat dorsal root ganglion.

We investigated the mRNA levels of interleukin-6-related genes in a rat dorsal root ganglion after application of a tourniquet to a hind limb in order to identify the molecules that are induced immediately after peripheral nerve injury at the early stage. Induction of interleukin-6 and upregulation of glycoprotein 130 mRNA expressions were observed in the ipsilateral dorsal root ganglion at 4 h after tourniquet application. Interleukin-6 protein was detected in small-sized and medium-sized dorsal root ganglion cells by immunohistochemical analysis. The induction of interleukin-6 expression is likely to play a role in the protection of injured neurons perhaps related to growth of their axons. Glycoprotein 130 might also account for the inhibitory effects following nerve injury.

Diagnostic Performance of Triagetrade mark for Benzodiazepines: Urine Analysis of the Dose of Therapeutic Cases.

We evaluated the diagnostic performance of Triage for benzodiazepines in 74 urine specimens from outpatients given therapeutic doses of benzodiazepines and compared the results of EMIT assays. Results obtained in all urine samples were confirmed using liquid chromatography-mass spectrometry (LC-MS). Overall agreement between results of Triage and EMIT assays was 73%. All of the Triage-positive samples were also positive by EMIT assays. Results of Triage and EMIT assays were different for 20 samples obtained from patients given thienodiazepines (etizolam, brotizolam, and clotiazepam) and nitrobenzodiazepines (nitrazepam, flunitrazepam, and clonazepam). LC-MS confirmed parent drugs in urine specimens, consistent with the prescriptions of drugs. The low agreement between Triage and EMIT results in this study might be due to low sensitivity of Triage for thienodiazepines. Thienodiazines are frequently prescribed benzodiazepines, and Triage panel is the most frequently used screening kit in Japan. It should be noted that negative results obtained by a Triage test might not mean the absence of thienodiazepines.

Medico-legal aspects of traumatic injury of the vertebrobasilar artery.

Based on our experiences with medico-legal autopsies, we analyzed traumatic injury of the vertebrobasilar artery (VBA) in traffic accident victims. VBA rupture occurred in six cases with traumatic medullary lesions, but in none with cervical cord injury. A relatively small external force applied to the head or neck can induce isolated traumatic subarachnoid hemorrhage (SAH), which accounts for approximately 11% of fatal head injuries. We examined the relationship between site of impact and site of vascular injury in 16 cases of isolated traumatic SAH. The results showed that the vertebral artery (VA) on the side of impact tended to be longitudinally injured in victims experiencing an external force equivalent to that of a fist punch to the head or face. There was no clear relationship between the site of impact and the site of vascular injury in victims who had sustained an external force greater than that of a fist punch. However, our results suggest that an external force causing rotation of the head was likely to cause tearing of the artery. In cases in which an external force equivalent to that of a fist punch was applied to the head, a raised blood alcohol level was a significant risk factor for artery rupture. Furthermore, a difference in the diameters of the left and right VAs was a risk factor for artery rupture. To determine why the VA, the most frequent site of arterial dissection, is also frequently the site of injury, we histologically examined normal and dissected VBAs. We also discuss medico-legal issues of the causal relationship between external force applied and rupture or dissection of the VA.

Diagnostic performance of Triage for benzodiazepines: urine analysis of the dose of therapeutic cases.

We evaluated the diagnostic performance of Triage for benzodiazepines in 74 urine specimens from outpatients given therapeutic doses of benzodiazepines and compared the results of EMIT assays. Results obtained in all urine samples were confirmed using liquid chromatography-mass spectrometry (LC-MS). Overall agreement between results of Triage and EMIT assays was 73%. All of the Triage-positive samples were also positive by EMIT assays. Results of Triage and EMIT assays were different for 20 samples obtained from patients given thienodiazepines (etizolam, brotizolam, and clotiazepam) and nitrobenzodiazepines (nitrazepam, flunitrazepam, and clonazepam). LC-MS confirmed parent drugs in urine specimens, consistent with the prescriptions of drugs. The low agreement between Triage and EMIT results in this study might be due to low sensitivity of Triage for thienodiazepines. Thienodiazines are frequently prescribed benzodiazepines, and Triage panel is the most frequently used screening kit in Japan. It should be noted that negative results obtained by a Triage test might not mean the absence of thienodiazepines.

Induction of nerve growth factor mRNA in a rat dorsal root ganglion after application of a tourniquet.

We investigated the mRNA levels of neurotrophins and neurotrophin receptors in a rat dorsal root ganglion (DRG), after tourniquet application to a hind limb, to identify the nerve-protective molecules that are induced immediately after peripheral nerve crush and play a part in the process leading to secondary events. No significant expression of nerve growth factor (NGF) mRNA or protein was observed in the control or contralateral DRG. NGF mRNA expression started within 2 h and NGF protein expression was observed in Schwann cells at 4 h after application of the tourniquet, due to termination of the neurotrophin supply from peripheral nerves. The levels of neurotrophin 3 mRNA were significantly increased in the DRGs on both sides at 1 and 2 h after tourniquet application, but no significant changes in brain-derived neurotrophic factor and neurotrophin 4/5 expression levels were observed in either the contralateral or ipsilateral DRG. The expression levels of neurotrophin receptors in the DRGs on both the contralateral and ipsilateral sides had decreased at 1 to 2 h after application of the tourniquet and had returned to the control levels at 4 h after tourniquet application.

[Lifetime social, psychological and physical background of suicides --research on the number of suicides during a year in Fukushima prefecture].

We performed a questionnaire survey of the bereaved families of decedents in 523 cases of suicide which occurred during the year beginning July 17, 1997, in Fukushima prefecture in Japan. The questionnaire consisted of 33 items which indicated decedents' behavior changes and stress factors before death. Of the 523 cases, 420 questionnaires were completed by the surviving family members. The risk factors for suicide which are conventionally accepted were found with high frequency in the data. Common stress factors were occupational and financial problems in males and difficulties with human relationships and family problems in females. The sexes were clearly differentiated in this respect. Common changes preceding suicide were expressed as "becoming depressed" or "social isolation". These statements were common and tended to be associated with depression. Hanging was the most frequent method of committing suicide in both sexes and at all ages. Thirty one percent of the victims were diagnosed and treated for mental diseases, and 39% for physical diseases. Many of the 39% who had physical diseases also showed depressive behaviors. The screening and treatment of depression is a matter of highest priority in the strategy of suicide prevention. It is therefore important that general practitioners, occupational physicians and public health nurses improve their skills in managing depressive disorder as a common condition. Psychiatrists should undertake their role of education in the diagnosis and treatment of depressive disorders more seriously.

Histological study of the normal vertebral artery--etiology of dissecting aneurysms.

Histological evaluation of dissecting aneurysms of the cerebral arteries has suggested that defects in the internal elastic lamina (IEL) induce dissection of the arterial wall. Dissecting aneurysms occur most frequently in the vertebral artery (VA). The present study examined sections of the normal VA to elucidate the mechanisms of arterial dissection. Bilateral VAs (20 vessels) were obtained from 10 patients who died of causes other than intracranial lesions. The VAs were detached from the VA union to the site 10 mm proximal from the point penetrating the dura mater. The VAs were cut at 5-mm intervals, and each segment was observed using modified Masson's trichrome staining for elastic fibers. The thickness of the media and adventitia significantly thinned after the origin of the posterior inferior cerebellar artery (PICA). IEL defects were observed at 35 sites in 11 vessels from six subjects. There was a high incidence of IEL defects in the extradural portion and near the origin of the PICA, areas frequently involved in arterial dissection. There was a high incidence of intimal thinning at areas of IEL defects (19 of 35), and thinning was particularly marked distal to the origin of the PICA. In the absence of intimal thickening, the vascular strength at the site of IEL defects may be reduced, which would promote the occurrence and progression of arterial dissection.

Nitric oxide synthase expressions in rat dorsal root ganglion after a hind limb tourniquet.

We investigated the mRNA levels of neuronal, inducible, endothelial nitric oxide synthases (nNOS, iNOS, eNOS) and tumor necrosis factor-alpha (TNF-alpha) in a rat dorsal root ganglion (DRG) after tourniquet application to a hind limb to identify molecules that trigger secondary events after peripheral nerve injury. Significantly high nNOS, iNOS mRNA and protein levels were observed in the ipsilateral DRGs 4 h after tourniquet application but not in the contralateral or control DRGs. The levels of TNF-alpha, an inducer of iNOS, were significantly increased in the ipsilateral DRGs 1 h after tourniquet application. Large amounts of NO might result in damage to the host cells and induce apotosis to eliminate damaged cells during the early stage of nerve injury.

[Novel findings from an animal tourniquet shock model].

This article is a review of our experimental results regarding the physiological statuses and roles of chemical mediators in tourniquet shock, and a novel phenomenon, modulation reflex, that is commonly observed in this shock model is discussed. In a rabbit with a tourniquet applied to a hind limb for 24 hrs, blood pressure (BP) gradually falls after release of the tourniquet, but the decline in BP stops when a tourniquet is again applied to the hind limb, indicating that shock mediators are attributed to the hind limb. The levels of dipeptides (anserine and carnosine) and lysosomes in blood samples as well as the levels of leukotrienes (LTD4 and LTE4) in blood and muscle samples from rabbits in tourniquet shock were elevated. However, injection of a large amount of a dipeptide into an ear vein of a rabbit did not reduce BP, suggesting that both peptides may not be directly related with reduction in BP of rabbits in tourniquet shock. Injection of a platelet-activating factor (PAF) antagonist into an ear vein resulted in slight elevation of BP and the elevated level was maintained for about 1 to 4 hrs during the period of decline in BP in tourniquet shock. As for interleukin-6 (IL-6), IL-6-deficient mice at young ages have a significantly greater blood volume than do wild-type mice without concomitant changes in body composition. Therefore, the role for IL-6 in the regulation of peripheral circulation may be to elevate, not reduce BP. In mice in tourniquet shock, superoxide (O2-) production is observed in skeletal muscle cells and these cells correspond to mitochondria-rich cells. However, RT-PCR of muscle samples showed no significant nitric oxide synthase (NOS) mRNA expression after tourniquet release. Pretreatment with NOS inhibitors before tourniquet release reduced O2- production in the skeletal muscle. These results indicate that O2- produced in muscle subjected to ischemia/repefusion may be involved in shock. As for changes in mRNA expression patterns of pro-inflammatory cytokines and nerve growth factors in blood samples from rats in tourniquet shock, up-regulation of M-CSF mRNA began at 2 h after tourniquet application and was short-lived. The level of ATF-3 mRNA had increased at 1 h and NGF mRNA gradually increased and reached a significantly high level at 4 h after tourniquet application. These results indicate that the transient mRNA expressions probably trigger secondary events that may be beneficial to wound repair and regeneration. In the early stage of tourniquet shock, the levels of IL-6 mRNA in the liver and kidneys of rats increased progressively and significantly, and the levels of iNOS mRNA in the kidneys increased. These findings suggest that that humoral and/or cellular mediators produced locally in the hind limb are responsible for remote organ injuries. Thus, these mediators, interacting each other, may contribute to the progress of shock. We have also found a novel phenomenon in tourniquet shock using rabbits. When a tourniquet is applied to the upper hind limb of a rabbit for 24 hrs, and pressure is applied to the femoral medial area immediately after tourniquet release, a reflex of decrease in blood pressure and decrease in heart rate, which last for a short period, is usually observed. This reflex is mediated through the ipsilateral femoral nerves, central nervous system and vagal nerves. Since the modulation reflex may be due to peripheral nerve injury, we investigated morphological and molecular changes in sciatic nerves and dorsal root ganglion (DRG) neurons in rats after tourniquet application. At 4 hr after tourniquet application, light microscopic examination showed only degeneration of the tourniquet segment in the sciatic nerve but no morphological changes in the DRG, while electron microscopic examination revealed mitochondrial swelling in some DRG neurons on the tourniquet-applied side and calcium deposition in these swollen mitochondria. These findings suggest that peripheral nerve injury induced a large amount of calcium influx into neuronal cell somas and that excess amounts of calcium-influx into neurons resulted in mitochondial swelling. Results of mRNA level analyses showed NGF mRNA expression followed by NGF protein expression in Schwann cells of the ipsilateral DRGs at 4 h after tourniquet application but not in the contralateral or control DRGs. Similarly, significantly high nNOS and iNOS mRNA levels were observed in the ipsilateral DRGs at 4 h, and expressions of nNOS and iNOS proteins were detected in the ganglion of the ipsilateral DRG. In addition, the TNF-alpha mRNA levels were significantly increased in the ipsilateral DRGs at 1 h after tourniquet application, indicating that TNF-alpha was activated in the early stage of nerve injury and then induced iNOS mRNA expression. Large amounts of nitric oxide (NO) produced by iNOS might result in damage to the host cells, and an overdose of NO might induce apoptosis and eliminate damaged cells during the early stage of nerve injury.