Experimental venous thrombi: MRI characteristics with histopathological correlation.

OBJECTIVES: The purpose of this study was to evaluate the MRI characteristics of venous thrombus over set time thresholds with histopathological correlation in a porcine model. METHODS: Inferior vena cava thrombi were induced in 12 pigs. MRI was performed in three pigs 2 h, 1 day, 3 days and 2 weeks after thrombus induction. RESULTS: The MRI characteristics were analysed in correlation with histopathological findings. The thrombi after 2 hours, which consisted of red blood cells (RBCs), showed isointensity on T(1 )weighted images (T(1)WIs) and hyperintensity on both T(2 )weighted images (T(2)WIs) and diffusion-weighted images (DWIs). The mean apparent diffusion coefficient (ADC) value was 1.93 × 10(-3) mm(2) s(-1). The thrombi after Day 1, which consisted of RBCs and migrating neutrophils at the periphery, showed isointensity on T(1)WIs, slight hyperintensity on T(2)WIs and hypointensity on DWIs. The mean ADC value was 1.62 × 10(-3) mm(2) s(-1) [corrected]. The thrombi after Day 3, which consisted of RBCs and peripheral inflammatory cells including macrophages, showed isointensity with peripheral hyperintense regions on T(1)WIs and hypointensity on both T(2)WIs and DWIs. The mean ADC value was 1.67 × 10(-3) mm(2) s(-1). After 2 weeks, the thrombi, which revealed RBC lysis surrounded by granulation tissues, showed isointensity on T(1)WIs and hyperintensity on T(2)WIs and DWIs. The mean ADC value was 2.48 × 10(-3) mm(2) s(-1). CONCLUSION: The temporal MRI characteristics seemed to be related to chemical and physical changes in RBC and organisation of granulation tissues. Free radicals generated by macrophages might also be related to some extent.

Quality of portal verification radiography using EC-L film in electron beam therapy.

The quality of the portal verification radiographs produced using the enhanced-contrast localisation (EC-L) Fast cassette-EC-L film (F-EC) combination and the EC-L Oncology cassette-EC-L film (O-EC) combination was investigated fundamentally and clinically. A computerised radiography (CR) system was used for comparison. In the clinic, portal verification radiographs produced for 22 patients with breast cancer were evaluated. The characteristic curves showed that the relative speed was 0.92 for the O-EC combination when the speed of the F-EC combination was defined to be 1, and that the average gradients were 4.76 and 4.35 for the F-EC combination and the O-EC combination, respectively. The smallest visible volumes of Burger's phantom were 50.3 mm(3), 60.8 mm(3) and 199.5 mm(3) for the F-EC combination, the O-EC combination and the CR system, respectively, at an energy of 9 MeV, and 68.4 mm(3), 74.2 mm(3) and 195 mm(3), respectively, at an energy of 12 MeV. In the clinic, both combinations at an energy of 6 MeV and the O-EC combination at 9 MeV showed very poor quality owing to underdensity. However the F-EC combination at an energy of 12 MeV and the O-EC combination at an energy of 15 MeV demonstrated a higher quality. When bremsstrahlung dose passing through the body is sufficient, the quality of portal verification radiography using EC-L film is appropriate for clinical use.

[Investigation of the local heating caused by a closed conducting loop at clinical MR imaging: Phantom study].

PURPOSE: Several reports have suggested that unusual thermal injuries in magnetic resonance (MR) imaging have occurred due to a closed conducting loop formed accidentally in a part of the patient's body. In this study, we investigated the relationship between the increases in temperature and several parameter settings for MR imaging by use of a human body-equivalent phantom. METHOD AND MATERIALS: A standard clinical 1.5T MR system (SIGNA HORIZON; GE) and a pelvic phased-array coil were used. The human body-equivalent phantom (agar, 0.9% saline, antiseptic) simulated a part of the pelvis and both femurs in a patient. A closed conducting loop could be reproduced when two ends of femurs contacted each other at a point, so that we could measure the temperature changes without and with a closed conducting loop. The temperature of the phantom was measured at the contact point of a closed conducting loop and the center of phantom by use of an optical fiber thermometer which was immune to the influences of radiofrequency (RF) and magnetic and electronic fields. We tested two imaging sequences of spin echo (SE) and fast spin echo (FSE) with 60 minutes of scanning time. In addition to the standard imaging sequences we measured temperature changes without the RF irradiation or gradient magnetic fields. The average temperature changes were recorded from five measurements which were repeated at intervals of more than one day. RESULTS: When the closed conducting loop was reproduced, the temperatures at the contact point significantly increased (p<0.001) compared with the temperatures at the center of phantom. The temperature changes at 60 minutes of scanning time were 7.0 and 8.1 degrees C by use of the SE and FSE, respectively. There were no significant temperature changes when the imaging was performed without the RF irradiation. CONCLUSION: Our result obtained by use of a human body-equivalent phantom demonstrated that local heating, which can lead to thermal injuries accidentally, could occur when a closed conducting loop was formed in part of the patient body. CLINICAL RELEVANCE/APPLICATION: Radiologists should be more careful about local heating which can occur in patients during clinical MR imaging by a closed conducting loop.

Involvement of vacuolar H+ -ATPase in incorporation of risedronate into osteoclasts.

Although osteoclasts incorporate bisphosphonates during bone resorption, the mechanism of this incorporation by osteoclasts is not known. We previously reported that bisphosphonates disrupt the actin rings (clear zones) formed in normal osteoclasts, but did not disrupt actin rings in osteoclasts derived from osteosclerotic oc/oc mice, which have a defect in the gene encoding vacuolar H(+)-ATPase (V-ATPase). The present study showed that V-ATPase is directly involved in the incorporation of risedronate, a nitrogen containing bisphosphonate, into osteoclasts. Treatment of osteoclasts with risedronate disrupted actin rings and inhibited pit formation by osteoclasts on dentine slices. Bafilomycin A(1), a V-ATPase inhibitor, inhibited the pit-forming activity of osteoclasts but did not disrupt actin rings. Risedronate failed to disrupt actin rings in the presence of bafilomycin A(1). E-64, a lysosomal cysteine proteinase inhibitor, showed no inhibitory effect on the demineralization of dentine by osteoclasts but inhibited the digestion of dentine matrix proteins without disrupting actin rings. Risedronate disrupted actin rings even in the presence of E-64. Treatment of osteoclasts placed on plastic plates with risedronate also disrupted actin rings. Bafilomycin A(1) but not E64 prevented the disruption of actin rings in osteoclasts treated with risedronate on plastic plates. Inhibition of V-ATPase with bafilomycin A(1) also prevented disruption of actin rings by etidronate, a non-nitrogen-containing bisphosphonate. These results suggest that V-ATPase induced acidification beneath the ruffled borders of osteoclasts and subsequent bone demineralization triggers the incorporation of both nitrogen-containing and non-nitrogen-containing bisphosphonates into osteoclasts.

Porcine enamel matrix derivative enhances trabecular bone regeneration during wound healing of injured rat femur.

To elucidate the effects of enamel matrix derivative (EMD: Emdogain) on bone regeneration in rat femurs after drill-hole injury, defects in bone were filled with either EMD or its carrier, PGA, as control. On postoperative days 4 to 28, dissected femurs were examined by means of various morphological approaches. In both experimental groups, formation of trabecular bone, which was immunostained for bone sialoproteins (BSP), had occurred in the medullary cavities at cylindrical bone defects on Day 7 postoperatively. Cuboidal osteoblasts were clearly observed on these newly-formed BSP-positive bone trabeculae. On Days 7 and 14, many multinucleated giant cells, which strongly expressed cathepsin K, had appeared on these bone trabeculae, indicating active bone remodeling. In these bone trabeculae, Ca and P weight % and Ca/P ratio were similar to those of cortical bone, and there was no significant difference between the PGA- and EMD-applied groups. Bone volume fraction of newly-formed bone trabeculae on Day 7 postoperatively was significantly higher in the EMD-applied group than in the PGA-applied controls. Because of active bone remodeling and the marked decrease of bone volume, on Days 14 and 28 postoperatively, however, there was no longer a significant difference in trabecular bone volume fraction between the experimental groups. Our results suggest that EMD possesses an osteo-promotive effect on bone and medullary regeneration during wound healing of injured long bones.

Effects of brefeldin-A: potent inhibitor of intracellular protein transport on ultrastructure and resorptive function of cultured osteoclasts.

Brefeldin-A (BFA) is a specific and potent inhibitor of the intracellular transport of clathlin-uncoated transitional vesicles from the cisterns of rough-surfaced endoplasmic reticulum (RER) to the Golgi lamellae. This study was designed to clarify the effects of BFA on ultrastructure, subcellular localization of vacuolar-type H+-ATPase and a lysosomal cysteine proteinase, cathepsin K, in cultured osteoclasts and their resorptive function. H+-ATPase and cathepsin K are the most important enzymes for decalcification of apatite crystals and degradation of type-I collagen, respectively. In control cultures without BFA, osteoclasts were structurally characterized by the development of broad ruffled borders and clear zones, and formed many resorption lacunae in cocultured dentine slices. In BFA-treated cultures, osteoclasts lacked ruffled borders, and the cytoplasm was filled with regular-size and extremely large pale vacuoles over 2 microm in diameter, which were produced by fusion of adjacent vacuoles. BFA did not, however, inhibit clear zone formation and adhesion of osteoclasts to dentine slices. Resorption lacuna formation was markedly diminished by BFA treatment. Although H+-ATPase and cathepsin K were strongly expressed in osteoclast ruffled borders in control cultures, BFA treatment altered the subcellular localization and decreased the expression of these molecules. In BFA-treated cultures, H+-ATPase immunoreaction in osteoclasts was observed along the limiting membranes of some, but not all, regular-size pale vacuoles, but neither in extremely large vacuoles nor along the smooth plasma membranes facing the dentine slices. Similarly, cathepsin K was localized within lysosomes and some regular-size pale vacuoles, but its secretion toward the dentine slices through the ruffled borders was strongly inhibited by BFA treatment. These results suggest that 1.) formation of the osteoclast ruffled borders and their resorptive function are closely associated with the intracellular transport of these molecules from the RER cisterns and the Golgi lamellae to the ruffled borders, and 2.) both H+-ATPase and cathepsin K are selectively transported to the ruffled border membranes by pale vacuoles.

cis-Acting elements responsible for low-temperature-inducible expression of the gene coding for the thermolabile isocitrate dehydrogenase isozyme of a psychrophilic bacterium, Vibrio sp. strain ABE-1.

Transcriptional control of the low-temperature-inducible icdII gene, encoding the thermolabile isocitrate dehydrogenase of a psychrophilic bacterium, Vibrio sp. strain ABE-1, was found to be mediated in part by a transcriptional silencer locating at nucleotide positions -560 to -526 upstream from the transcription start site of icdII. Deletion of the silencer resulted in a 20-fold-increased level of expression of the gene at low temperature (15 degrees C) but not at high temperature (37 degrees C). In addition, a CCAAT sequence located 2 bases upstream of the -35 region was found to be essential for the low-temperature-inducible expression of the gene. By deletion of this sequence, low-temperature-dependent expression of the gene was completely abolished. The ability of the icdII promoter to control the expression of other genes was confirmed by using a fusion gene containing the icdII promoter region and the promoterless icdI open reading frame, which encodes the non-cold-inducible isocitrate dehydrogenase isozyme of Vibrio sp. strain ABE-1. Escherichia coli transformants harboring icdII acquired an ability to grow rapidly at low temperature.

Effect of lactic acid on water content and osmotic fragility of erythrocytes in vitro.

Effects of lactic acid in red blood cells on osmotic fragility and water content of erythrocytes after hyperthermia were investigated. The osmotic fragility of erythrocytes increased following one-hour incubation with the addition of lactic acid at both 37 degrees C and 42 degrees C and that also increased after heating in vitro at 42 degrees C compared with those incubated at 37 degrees C, whether the lactic acid was added or not. The water content increased with the addition of lactic acid after heating in vitro at 42 degrees C. A high concentration of lactic acid and hyperthermia seem to cause the increase of intracellular water and the decrease of osmotic resistance of the red blood cells.

Differential expression in Escherichia coli of the Vibrio sp. strain ABE-1 icdI and icdII genes encoding structurally different isocitrate dehydrogenase isozymes.

The expression of two structurally different isocitrate dehydrogenase isozymes of Vibrio sp. strain ABE-1 in Escherichia coli was examined. At a low temperature (15 degrees C), a thermolabile and monomeric type isozyme (IDH-II), which is quite different in amino acid sequence from the E. coli isocitrate dehydrogenase, was expressed and conferred glutamate prototrophic ability on an E. coli mutant defective in isocitrate dehydrogenase. The ability of IDH-II to confer restoration of the E. coli mutant to glutamate prototrophy was similar to that of IDH-I, which is a dimeric enzyme homologous to the E. coli isocitrate dehydrogenase. At a high temperature (37 degrees C), no functional IDH-II was expressed. Transcription of icdI and icdII genes, which encode IDH-I and IDH-II, respectively, was regulated differently by different environmental conditions. The level of icdII mRNA was increased by lowering the growth temperature for E. coli transformants, while the level of icdI mRNA was increased when E. coli transformants were cultured in acetate minimal medium. Similar patterns of transcriptional regulation of the two icd gene were observed also in Vibrio sp. strain ABE-1. However, activity of isocitrate dehydrogenase kinase, which can phosphorylate IDH-I and consequently inactivate the enzymatic activity, was detected in cell lysates of E. coli but not of Vibrio sp. strain ABE-1.

Genes encoding two isocitrate dehydrogenase isozymes of a psychrophilic bacterium, Vibrio sp. strain ABE-1.

The genes coding for two structurally different isocitrate dehydrogenase isozymes (IDH-I and IDH-II) of a psychrophilic bacterium, Vibrio sp. strain ABE-1, were cloned and sequenced. Open reading frames of the genes (icdI and icdII) are 1,248 and 2,229 bp in length, respectively. The amino acid sequences predicted from the open reading frames of icdI and icdII corresponded to the N-terminal amino acid sequences of the purified IDH-I and IDH-II, respectively. No homology was found between the deduced amino acid sequences of the isozymes; however, the IDH-I, a dimeric enzyme, had a high amino acid sequence identity (74.3%) to the Escherichia coli IDH. The deduced amino acid sequence of the IDH-II, a monomeric enzyme, was not related to any known sequence. However, the IDH-II had an amino acid sequence homologous to that of a cyanogen bromide-cleaved peptide containing a putative active-site methionyl residue of the monomeric IDH of Azotobacter vinelandii. The two genes (icdlI and icdII) were found to be tandemly located in the same orientation. Northern (RNA) blot analyses showed that the two genes are transcribed independently. Primer extension experiments located single transcriptional start sites 39 and 96 bp upstream of the start codons of icdI and icdII, respectively. The amount of icdI transcript but not icdII increased when Vibrio sp. strain ABE-1 cells were cultured in acetate minimal medium.

Purification and characterization of monomeric isocitrate dehydrogenase with NADP(+)-specificity from Vibrio parahaemolyticus Y-4.

NADP(+)-dependent isocitrate dehydrogenase [IDH: EC 1.1.1.42] was purified to electrophoretic homogeneity from Vibrio parahaemolyticus Y-4, and shown to be a monomeric protein of molecular weight 80,000 with a pI of 5.0. The amino acid composition and partial sequence at the N-terminus resembled those reported for other bacterial monomeric IDHs. Immunotitration with antisera to the monomeric and dimeric enzymes (antisera to IDH-II and -I of Vibrio ABE-1) showed an immunochemical distinction between the monomeric and dimeric IDHs, but there is similarity within the IDHs of each group. The circular dichroism spectra of the native and heat-denatured enzyme are also similar to those of monomeric IDH (IDH-II of Vibrio ABE-1). These monomeric IDHs are proteins comprising 17-22% helix and 25-35% beta-pleated sheet in the native state.

MK-801 prevents the post-ischemic cerebral hypoperfusion, but not the dysfunction of the vagal baroreflex in dogs.

Pretreatment with MK-801, a non-competitive N-methyl-D-aspartate (NMDA) antagonist, failed to protect the vagal component of reflex bradycardia from 5-min global cerebral ischemia in dogs under pentobarbital anesthesia. On the other hand, MK-801 completely prevented the development of the post-ischemic cerebral hypoperfusion without affecting the cerebral blood flow in sham-operated animals. The results suggest that NMDA receptors may participate in the development of the secondary disturbance of the cerebral circulation, but are not involved in the post-ischemic dysfunction of the baroreflex system.

Selective dysfunction of the vagal component of the baroreflex following cerebral ischemia: protection by ifenprodil and flunarizine.

Baroreflex sensitivity assessed from the phenylephrine-induced reflex bradycardia was significantly decreased following 5 min global incomplete cerebral ischemia in pentobarbitalized dogs. Although bilateral vagotomy in the cervical region decreased baroreflex sensitivity by about 50% in sham-operated animals, it hardly affected the baroreflex in animals subjected to ischemia. The extent of the decrease in the influence of vagotomy on the baroreflex was dependent on the severity of ischemia in the dorsal medulla oblongata. In animals vagotomized before ischemia, no significant decrease in baroreflex sensitivity was observed following ischemia. Pretreatment with ifenprodil or flunarizine, 1 mg/kg i.v., 5 min prior to ischemia prevented the post-ischemic decrease in baroreflex sensitivity. Vagotomy decreased baroreflex sensitivity during the reperfusion period in these treated animals. These results suggest that the post-ischemic attenuation of reflex bradycardia may be due to a selective dysfunction of the vagal component of baroreflex, which can be prevented by the cerebroprotective agents.

Protective effect of beraprost sodium, a new chemically stable prostacyclin analogue, against the deterioration of baroreceptor reflex following transient global cerebral ischaemia in dogs.

1. A possible cerebroprotective effect of a chemically stable prostacyclin analogue, beraprost sodium, was investigated in a canine model of cerebral ischaemia. Cerebral ischaemia was produced by the combined occlusions of the left subclavian and the brachiocephalic arteries with preceding ligations of the intercostal arteries. 2. The decrease in baroreceptor reflex sensitivity (BRS), measured by phenylephrine-induced reflex bradycardia, following 5 min ischaemia was used to assess the cerebroprotective effect. 3. Beraprost (1 microgram kg-1 min-1 i.v., infused for 15 min just before ischaemia) completely prevented the decrease in BRS. Although the lower dose of beraprost (0.1 microgram kg-1 min-1 i.v.) failed to show such a protective effect, its inhibitory effect on ADP-induced platelet aggregation was as potent as that of the higher dose. 4. The extent of decrease in BRS was inversely correlated with the extent of the residual blood flow in the medulla oblongata during ischaemia. Since beraprost did not affect the extent of the residual blood flow during ischaemia, its cerebroprotective effect could not be ascribed to the reduction of the degree of ischaemia by increasing collateral blood flow to the brain. 5. Post-ischaemic reduction of the regional blood flow in the medulla and the cerebral cortex was completely prevented by the higher dose of beraprost. 6. The present study suggests that the cerebroprotective effect of beraprost may be independent of its anti-aggregatory and vasodilator effects. It is possible that the protection may be due to a prostacyclin-like cytoprotective effect through membrane stabilization.

Deterioration of baroreflex by transient global cerebral ischemia: its correlation with the degree of ischemia or post-ischemic hypoperfusion in the medulla oblongata.

In a canine model of transient global cerebral ischemia, the correlation between the decrease in baroreflex sensitivity (BRS) following 5-min ischemia and the degree of ischemia or post-ischemic hypoperfusion was investigated. Although the medulla oblongata and the cerebral cortex suffered a similar degree of ischemia, the extent of post-ischemic decrease in BRS was inversely correlated with the residual blood flow during ischemia in the medulla, but not with that in the cerebral cortex. A similar degree of post-ischemic hypoperfusion occurred in the medulla and the cerebral cortex. However, the extent of decrease in BRS was not correlated with the degree of hypoperfusion, and the cortical EEG was not significantly affected. These results suggest that the decrease in BRS may be due to the functional damage in the medulla and that the selective decrease in BRS without concomitant impairment of the EEG cannot be ascribed to the regional difference in the degree of ischemia or post-ischemic hypoperfusion.