The novel calpain inhibitor SJA6017 improves functional outcome after delayed administration in a mouse model of diffuse brain injury.

A principal mechanism of calcium-mediated neuronal injury is the activation of neutral proteases known as calpains. Proteolytic substrates for calpain include receptor and cytoskeletal proteins, signal transduction enzymes and transcription factors. Recently, calpain inhibitors have been shown to provide benefit in rat models of focal head injury and focal cerebral ischemia. The present study sought to investigate, in experiment 1, the time course of calpain-mediated cytoskeletal injury in a mouse model of diffuse head injury by measuring the 150- and 145-kDa alpha-spectrin breakdown products (SBDP). Secondly, in experiment 2, we examined the effect of early (20 min postinjury) administration of the novel calpain inhibitor SJA6017 on functional outcome measured 24 h following injury and its effect on posttraumatic alpha-spectrin degradation. Lastly, in experiment 3, we examined the effect of delayed (4 or 6 h postinjury) administration of SJA6017 on 24-h postinjury functional outcome. In experiment 1, isoflurane-anesthetized male CF-1 mice (18-22 g) were subjected to a 750 g-cm weight drop-induced injury and were sacrificed for SBDP analysis at postinjury times of 30 min, and 1, 2, 6, 24 and 48 h (plus sham). In experiments 2 and 3, mice were injured as described, and delivered a single tail vein injection of either SJA6017 (0.3, 1, or 3 mg/kg) or vehicle (administered immediately, 4 or 6 h postinjury [3 mg/kg]). Functional outcome was evaluated in both studies, and, in experiment 2, 24-h postinjury assessment of SBDPs was determined. Following injury, the level of SBDP 145 was significantly different from sham at 24 and 48 h in cortical and at 24 h in the hippocampal tissues and at 48 h in the striatum. Immediate postinjury administration of SJA6017 resulted in a dose-related improvement in 24-h functional outcome (p < 0.05 at 3 mg/kg). Significance was maintained after a 4-h delay of the 3 mg/kg, but was lost after a 6-h delay. Despite improvement in functional outcome at 24 h, SJA6017 did not reduce spectrin breakdown in cortical or hippocampal tissues. These results support a role for calpain-mediated neuronal injury and the potential for a practical therapeutic window for calpain inhibition following traumatic brain injury. However, measurements of regional spectrin degradation may not be the most sensitive marker for determining the effects of calpain inhibition.

Visualization of central stellate fibrosis in hyaline vascular type Castleman's disease.

Hyaline vascular type Castleman's disease (HVCD) is a benign cause of lymph node hypertrophy which radiologically resembles tumours such as paraganglioma and lymphoma. However, a distinguishing pathological characteristic of HVCD is the presence of central stellate fibrosis. Since conventional CT and MRI scans failed to differentiate areas of fibrosis, three-phase dynamic CT was performed on a patient who was thought to have HVCD. The images showed central fibrosis and consequently the correct pre-operative diagnosis of HVCD was made.

A new strategy for management of retroperitoneal tumors with supradiaphragmatic vena caval thrombi.

A new surgical technique to treat retroperitoneal tumors with supradiaphragmatic vena caval invasion is described. In this technique, hepatic warm ischemia can be avoided with reversed hepatic outflow through the portal vein and neither hypothermic circulatory arrest nor cardiopulmonary bypass is necessary using centrifugal blood pump-driven bypass.

Difference in onset of warm ischemia and reperfusion injury between parenchymal and endothelial cells of the liver. Evaluation by purine nucleoside phosphorylase and hyaluronic acid.

The onset of warm ischemia and reperfusion injury in the liver was investigated in a canine model through changes in parenchymal markers [isozyme class V of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT)], endothelial markers [purine nucleoside phosphorylase (PNP) and hyaluronic acid clearance], and the liver metabolism (ketone body ratio) in warm ischemia induced by inflow occlusion using Pringle's maneuver and subsequent reperfusion. In this in vivo model, a PNP assay system and a model were designed so as to exclude the influence of wide localization of PNP possibly originating in erythrocytes or the intestine, and to discriminate between PNP of endothelial cells and that of parenchymal cells in the liver. After 45 min of warm ischemia, reperfusion resulted in damage only to endothelial cells, as seen by significant increase in PNP alone (3.6 +/- 0.1 U/liter at the end of warm ischemia to 6.8 +/- 0.5 U/liter at 5 min after reperfusion, P < 0.01) and significant decrease in hyaluronic acid clearance compared to the 30-min warm ischemia group in which no increase in either marker for parenchymal and endothelial cells was noted. By contrast, after 60 min of warm ischemia, reperfusion resulted in damage to parenchymal cells along with damage to endothelial cells, as seen by significant increases in LDH(V) and ALT (93 +/- 4 U/liter and 32 +/- 2 IU/liter at the end of warm ischemia to 239 +/- 17 U/liter and 165 +/- 27 IU/liter at 5 min after reperfusion, respectively), as well as a marked increase in PNP and deterioration of hyaluronic acid clearance compared to the 45-min warm ischemia group. Reperfusion after 120 min of warm ischemia did not show recovery of metabolic function of the liver as evaluated by hepatic mitochondrial redox state. It is suggested that a time lag occurs in the onset of injury between parenchymal cells and endothelial cells and that endothelial cells are temporally earlier in failing than parenchymal cells when the liver is exposed to short-term warm ischemia and subsequent reperfusion.

Evaluation of the protective effect of a novel prostacyclin analog on mesenteric circulation following warm ischemia.

The protective effect of a novel prostacyclin (PGI2) analog, OP-2507, on mesenteric circulation was investigated in a canine warm ischemia model. In 20 mongrel dogs, the entire portion of the intestine supplied by the superior mesenteric artery (SMA) and drained by the superior mesenteric vein (SMV) was completely isolated, maintaining the blood and lymph vessels intact. Sixty or 120 min of complete warm ischemia (WI) of the intestine was induced by clamping SMA and SMV, followed by reperfusion for 120 min. Animals were divided into five groups (each n = 4): group 1, sham operation; group 2, 60 min WI; group 3, 120 min WI; group 4, 60 min WI with PGI2 analog administration; group 5, 120 min WI with PGI2 analog administration. The analog was administered at a rate of 6 micrograms.kg-1.h-1 immediately after laparotomy until the end of the observation period. Mean arterial pressure, SMA blood flow (SMABF), SMV pressure were monitored and total mesenteric vascular resistance (TMVR) was calculated. To evaluate the endothelial activation, endothelin, which is secreted from the endothelium under hypoxic stress, was assayed from blood samples of SMV. None of the animals showed significant changes in mean arterial pressure. In groups 2 and 3, SMABF decreased significantly to less than 60% of preoperative value (15 ml.kg-1.min-1) and TMVR significantly increased from 8.1 and 7.3 mm Hg.ml-1.kg.min before WI to 14.0 and 16.4 mm Hg.ml-1.kg.min after 120 min reperfusion, respectively, resulting in delayed hypoperfusion. In contrast, in groups 4 and 5, SMABF increased to over 100% of preoperative level, while TMVR declined from 7.8 and 8.4 mm Hg.ml-1.kg.min before WI to 6.2 and 6.3 mm Hg.ml-1.kg.min after 120 min reperfusion. After 60 min reperfusion, SMABF and TMVR showed a significant difference between the treated and nontreated groups. Only in group 3, high endothelin concentrations (over 20 pg/ml) were observed even after 120 min reperfusion. It was concluded that the PGI2 analog was able to suppress the endothelial activation and the disturbance of mesenteric circulation caused by WI and reperfusion.

Surgical approach for maintaining nonischemic conditions of the liver in acute hepatic vein obstruction.

To establish a possible surgical approach for preventing warm ischemic injury to the liver followed by hepatic vein occlusion (HVO), the hepatic hemodynamics and energy metabolism were investigated in an acute canine HVO model with and without hepatic arterial blood flow. Arterial blood ketone body ratio (AKBR; acetoacetate/3-hydroxybutyrate) and adenylate energy charge potential (ECP = [ATP + 1/2 ADP]/[ATP+ADP+AMP]) of the liver tissue were measured during and after 60 min of HVO. In the group with hepatic arterial blood flow, in which arterial blood was drained by hepatofugal portal flow via the venovenous bypass, total hepatic blood flow, portal vein pressure, ECP, and AKBR were maintained at almost normal level after the termination of HVO, resulting in the survival of all animals for 3 days or longer. By contrast, in the group without hepatic arterial blood flow (warm ischemic group), total hepatic blood flow was maintained at less than 60% of preischemic value, and portal vein pressure gradually increased up to twice the preischemic value. ECP decreased from 0.81 +/- 0.06 to 0.71 +/- 0.07 along with increasing portal venous pressure, and AKBR also decreased from 1.23 +/- 0.12 to 0.63 +/- 0.23, resulting in no survival longer 6 hr. It was concluded that hepatic arterial blood flow during HVO, if drained as hepatofugal portal flow, could maintain nonischemic conditions in the liver, despite vena cava obstruction, by providing an alternate outflow via reversed flow in the portal vein.

A new experimental model of specific liver hypoxia using membrane oxygenator.

The present study introduces a new experimental canine model of hepatic arterial deoxygenation using a membrane oxygenator to investigate the influence of hepatic arterial hypoxia on hepatic hemodynamics and energy metabolism. Eighteen mongrel dogs weighing 10 kg each were randomly divided into three groups: group A served as a control (118.0 +/- 9.0 mmHg of hepatic arterial O2 content), group B as a moderately deoxygenated group (40 mmHg of hepatic arterial O2 content), and group C as a severely deoxygenated group (25 mmHg of hepatic arterial O2 content). Deoxygenation was achieved by perfusion of a gas mixture of O2 and N2 through the membrane oxygenator, which was interposed between the femoral artery and the proper hepatic artery, for 60 min. In group C, hypoxia decreased the mean systemic arterial blood pressure and hepatic arterial blood flow. Arterial blood ketone body ratio (AKBR = acetoacetate/3-hydroxybutyrate), which reflects the hepatic mitochondrial redox state, rapidly decreased prior to the significant increase of glutamate oxaloacetate transminase, glutamate pyruvate transminase, and lactate dehydrogenase after the initiation of hypoxia. Hepatic arterial deoxygenation to 25 mmHg for 60 min induced injury to hepatic hemodynamics, resulting in the deterioration of systemic hemodynamics even after the termination of liver hypoxia. This in vivo temporal hepatic arterial hypoxic model without alteration of inflow volume might be useful for investigating the mechanism of hypoxic injury and the critical point of liver hypoxia on hepatic and/or systemic hemodynamics and liver viability.

Advantageous effect of low-molecular-weight heparin administration on hepatic mitochondrial redox state.

The effect of low-molecular-weight heparin (LMWH) on hepatic mithocondrial metabolism was compared with that of unfractionated heparin (UH) after the intravenous administration of these two kinds of heparin to normal rabbits. The magnitude of decrease in blood triglyceride levels 5 min after administration of UH (200 U/kg) was significantly greater than after LMWH (200 U/kg). Free fatty acid levels in the blood were significantly higher after this dose of UH than after LMWH. Blood total ketone body levels (acetoacetate + 3-hydroxybutyrate) 15 min after injection of 50 U/kg of UH were significantly higher than those after a dose of 50 U/kg of LMWH, and levels after 200 U/kg of UH were significantly higher than those after 200 U/kg of LMWH at 15, 30, 45 and 60 min. Enhanced ketogenesis was not noted after LMWH at any of the doses, or after UH at 3 U/kg. Arterial ketone body ratio (AKBR; acetoacetate/3-hydroxybutyrate), which reflects the hepatic mitochondrial oxidation-reduction state (NAD+/NADH), was maintained above 1.0 in all groups except in the U-200 group, while AKBR in that group was significantly decreased to 0.99 +/- 0.14 at 30 min, and further decreased to 0.80 +/- 0.08 at 60 min. These results indicate that LMWH has less effect on lipolysis than UH and does not enhance ketogenesis, resulting in less deterioration of mitochondrial redox state.

Hemodynamics and hepatic energy metabolism in canine model of acute hepatic venous occlusion with mesocaval shunt.

The relationship between portal hemodynamics and the energy metabolism of the liver with acute hepatic venous occlusion (HVO) was investigated by assessing the changes in the hepatic blood flow, arterial blood ketone body ratio (AKBR) and adenylate energy charge potential (ECP) of the liver tissue in canine model. Acute HVO was induced by the ligation of both the supra- and infrahepatic inferior vena cava (IVC) over the protruding ends of a heparin-coated polyethylene cannula inserted into the IVC. All dogs with only HVO (n = 5) died within 30 min. HVO dogs with additional mesocaval (MC) shunt (n = 10) survived longer than 7 days, during which time their AKBR were maintained in the normal range (over 1.0). ECP was also maintained above the normal level (over 0.850) during the 28-day period. Along with increasing portal pressure caused by the narrowing of the shunt anastomosis, the hepatic blood flow decrease gradually, resulting in a sudden decrease in AKBR and ECP when the portal pressure increased over 11 mm Hg. It is suggested that the normalization of portal pressure is one of the most important factors for maintaining the hepatic energy metabolism and that MC shunt is an effective therapy for maintaining the function of the liver with HVO, as long as portal pressure can be kept within normal range.

Preserved mitochondrial function by allopurinol despite deteriorated hemodynamics in warm ischemia-damaged canine liver.

To investigate the pathophysiology of warm ischemia (WI) of the liver, the changes in hemodynamics and energy metabolism were studied during and after 60-min complete WI induced by total hepatic vascular exclusion (HVE) in the canine model. Hepatic arterial blood flow after WI was maintained at 76% of the pre-ischemic level, while portal blood flow was only 27% of the pre-ischemic level associated with increased portal vein pressure, which was twice the pre-ischemic level, resulting in a decrease of total hepatic blood flow to 46% of the pre-ischemic level. Concentration of tissue lipid peroxide increased after WI. Arterial blood ketone body ratio (AKBR), which reflects the hepatic mitochondrial redox state, could not recover to the pre-ischemic level after termination of WI. However, when 100 mg/kg of allopurinol (xanthine oxidase inhibitor) was administered intravenously 10 min prior to initiating WI, AKBR was restored to the pre-ischemic level at 30 min after WI in spite of the fact that allopurinol administration to one group produced no remarkable changes in the hepatic hemodynamics compared with the group without allopurinol treatment. Concentration of adenine nucleotides was significantly higher for the treated group at the end of and after WI than for the group without allopurinol treatment and was maintained at a higher level even after WI. Lipid peroxide production was suppressed. Electron microscopic examination revealed that allopurinol treatment could not prevent mitochondrial swelling. It is suggested that WI causes injury primarily to the portal sinusoidal circulation, resulting in portal congestion concomitant with high portal pressure after the release of WI. Allopurinol could prevent the deterioration of mitochondrial ATP metabolism, and was able to inhibit lipid peroxide production, resulting in the rapid recovery of mitochondrial redox state in spite of the fact that it produced no amelioration of hepatic hemodynamics and morphological alterations.