OBJECTIVES: Osteoarthritis (OA) is a common joint deterioration initiated by multiple factors. To better understand related factors in the development of this disease, we focused on the mechanical stress loaded on articular cartilage. MATERIALS AND METHODS: The anterior cruciate ligaments of rabbit knee joints were transected, and expression of protein kinase C (PKC) examined immunohistochemically. The PKC activator 12-o-tetradecanoyl-phorbol-13-acetate (TPA) was then administered intraarticularly. To determine the involvement of gas mediators, a cartilage defect was made on the medical femoral condyle of rabbit knee joints. Hydrostatic pressure was loaded on the cartilage taken from the surrounding defects, and levels of superoxide anion and nitric oxide (NO) were measured. Bovine chondrocytes were subjected to cyclic mechanical stretch using a Flexercell Strain Instrument. Proteoglycan synthesis and PKC activity were measured. Expression of matrix metalloproteinase (MMP)-3 and tissue inhibitor of metalloproteinase (TIMP)-1 in articular cartilages obtained from OA patients were examined using Northern blots. RESULTS: Chondrocytes from experimentally induced OA were stained positively with anti-alpha-PKC antibody. Intraarticular administration of TPA prevented the development of OA changes. Cyclic tensile stretch loaded on chondrocytes decreased proteoglycan synthesis and PKC activity. Thus, PKC is involved in the stress-mediated degradation of articular cartilage. Cartilage defects led to degradation of surrounding cartilage and to enhanced superoxide anion and NO synthesis. We also noted increased and decreased expressions of MMP-3 and TIMP-1 mRNA in human OA cartilage, respectively. CONCLUSION: PKC, gas mediators (superoxide anion, NO), and proteinases are all involved in OA.

Animals↗

PubMed1998

Expression of Nedd2/ICH-1 (caspase-2) in the developing rat retina.

Expression and localization of Nedd2/ICH-1 (caspase-2) were examined in the developing rat retina. The expression of Nedd2 mRNA was conspicuous in the prenatal period and down-regulated in the postnatal period. In contrast, the expression of an alternatively spliced form of Nedd2 encoding a truncated protein, which has been shown to protect against programed cell death (PCD), was faint in the prenatal period but increased gradually in the postnatal period. Nedd2 immunoreactivity was observed in almost all cells of the ganglion cell layer (GCL) during the perinatal period, when some nuclei in the GCL showed pyknosis or fragmentation. Nedd2-positive cells in the neuroblastic layer (NBL) were present in patches at embryonic day 19, and began to assume a stratified arrangement at postnatal day 3, when nuclei showing PCD were observed in places across the NBL. At postnatal day 14, only faint Nedd2 immunoreactivity was observed in the GCL and the inner nuclear layer, and no Nedd2 immunoreactivity was present in the outer nuclear layer. These findings demonstrate the cell type- and differentiation-specific patterns of the Nedd2 expression in the developing retina.

Alternative Splicing↗

PubMed1998

Objective tinnitus caused by an aberrant internal carotid artery.

We report the case of a 29 year-old woman who complained of pulsatile tinnitus and impaired hearing. On otoscopic examination, her right tympanic membrane was observed to be in contact with a mass in the middle ear cavity, with the formation of a meniscus at the point of contact. Using a high-sensitivity microphone inserted into the external auditory canal, we recorded pulsatile tinnitus that was synchronous with the electrocardiogram. Magnetic resonance angiography revealed that the middle ear mass was an aberrant internal carotid artery coursing through the hypotympanum.

Adult↗

PubMed1998

Three-vessel occlusion using a micro-clip for the proximal left middle cerebral artery produces a reliable neocortical infarct in rats.

We demonstrate a new three-vessel occlusion model of temporary focal ischemia in Sprague-Dawley rats (n=13). Under a surgical microscope, bilateral carotid arteries and the left middle cerebral artery (MCA) were occluded for 2 h using a snare for the neck and a micro-clip for the MCA. The reduction of regional cerebral blood flow (rCBF) monitored by laser Doppler flowmetry was 28% on average compared to the preischemic control value (100%) during ischemia. At that level of CBF reduction, there was no thrombus formation in the proximal MCA trunk, and a full recovery of rCBF was confirmed in the ischemic core within 5 min of reflow. Two days later, the cerebral infarct was limited to the neocortex, and the size and variability of the infarct are considered feasible for the assessment of any therapeutic challenge against the ischemic damage due to temporary focal ischemia in this model.

Animals↗

PubMed1998

Stimulation of leukaemic cells from adult T-cell leukaemia patients with bacterial superantigens.

Bacterial superantigens stimulate T cells in a manner that is restricted to the Vbeta of the T-cell receptor. We examined the ability of adult T-cell leukaemia (ATL) cells to respond to these superantigens. Mononuclear cells from 10 patients were cultured with staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB) or toxic shock syndrome toxin-1 (TSST-1), and their response was determined by MTT assay and 3H-thymidine incorporation assay. Cells from six patients showed a specific response to a single superantigen. In two cases the cells responded to TSST-1 and bore Vbeta2, the known target of TSST-1. In three cases the cells responded to SEA with one bearing Vbeta9, a target of SEA, and one bearing Vbeta16. In one case the cells responded to SEB. Most of the cells which proliferated in response to superantigens were determined genetically to be leukaemic. The response to TSST-1 was inhibited by anti-Vbeta2 antibody. The responding cells showed a strongly enhancement expression of interleukin-2 receptor. These findings indicate that leukaemic cells from a proportion of ATL patients have an ability to respond to T-cell receptor-dependent superantigens. This suggests that bacterial infection in such patients may contribute to the expansion of ATL cells.

Adult↗

PubMed1998

Prevention of pulmonary embolization during excision of an infected venous thrombus.

We describe the case of a 64-year-old woman with an iatrogenic pseudoaneurysm, arteriovenous fistula, and thrombotic occlusion of the right femoral vein with Staphylococcus aureus infection resistant to methicillin as complications of femoral catheterization for hemodialysis access. A temporary caval filter was introduced just prior to operation and kept in place for 4 days. An infected thrombus was captured and removed with a filter-catheter device. The placement of a temporary caval filter should be considered for prevention of pulmonary embolism in patients with infected venous thrombosis which requires surgical thrombectomy.

Catheterization, Peripheral↗

PubMed1998

Nitric oxide synthase immunoreactivity related to cold-induced brain edema.

To determine the relationship between brain edema and the expression of nitric oxide synthase (NOS), we immunohistochemically studied the distribution and level of NOS in rat brain cold injury model. Vasogenic brain edema was produced by cortical freezing lesion. NOS immunohistochemical studies were performed 4 and 8 h, 1, 3, 5, 7, 14 and 21 days after injury. In control normotensive rats, immunoreactivity for NOS was observed in scattered neuronal cells as reported previously, but there was no reactivity in glial cells. In the present study in the cold injury model, however, fibrinogen staining showed extravasated plasma fluid extending to the white matter contralateral to the site of cold injury. NOS immunoreactivity was observed in most reactive astrocytes and a proportion of the microglial cells and macrophages in the white matter not only just beneath the area of cold injury but also in the contralateral side. The nerve cells in the edematous region scarcely showed additional immunoreactivity for NOS. The distribution of increased NOS relatively corresponded with the sites of extravasated plasma fluid demonstrated by fibrinogen staining. Electron microscopically, NOS was observed in astrocytes along the rough endoplasmic reticulum suggesting that NOS was produced in the cells and not taken up from the surroundings. Based on these findings, we postulate that brain edema and the simultaneously generated free radicals or some extravasated plasma components may induce expression of NOS in the reactive cells, and that the NO thus generated may be involved in the development of diffuse degeneration of the white matter which accompanies brain edema.

Animals↗

PubMed1998

Redox control of neuronal damage during brain ischemia after middle cerebral artery occlusion in the rat: immunohistochemical and hybridization studies of thioredoxin.

Thioredoxin (TRX) is a small, multifunctional protein with a redox-active site and multiple biological functions that include reducing activity for reactive oxygen intermediates. We assayed TRX and TRX mRNA by immunohistochemical methods and hybridization experiments in the rat brain after middle cerebral artery (MCA) occlusion. During ischemia, the immunoreactivity for TRX decreased; it disappeared after MCA occlusion in the ischemic regions. It rapidly decreased and nearly disappeared at 4 and 16 hours after MCA occlusion in the lateral striatum and frontoparietal cortex, respectively. On the other hand, in the perifocal ischemic region, the penumbra, TRX immunoreactivity began to increase 4 hours after MCA occlusion and continued to increase until 24 hours after occlusion. In hybridization experiments, TRX mRNA decreased and nearly disappeared 4 hours after MCA occlusion in the lateral striatum. In the frontoparietal cortex, it decreased until 24 hours after MCA occlusion. In the perifocal ischemic region, TRX mRNA began to increase 4 hours after MCA occlusion and continued to increase until 24 hours. Northern blot analysis showed that total TRX mRNA in the operated hemispheres was induced from 8 hours and increased until 24 hours after the surgical procedures. We previously reported that recombinant TRX promotes the in vitro survival of primary cultured neurons. We now suggest that TRX in the penumbra has neuroprotective functions and that decreased levels of TRX in the ischemic core modify neuronal damage during focal brain ischemia.

Animals↗

PubMed1998

Enhanced poly(ADP-ribosyl)ation after focal ischemia in rat brain.

Nitric oxide from neuronal cells plays detrimental roles in glutamate neurotoxicity and in focal brain ischemia. Nitric oxide directly damages DNA, and breaks in the DNA strands activate poly(ADP-ribose) polymerase (PARP), which brings poly(ADP-ribosyl)ation of the nuclear proteins. The excessive activation of PARP is thought to cause depletion of ATP and the energy failure resulting in cell death. To clarify the involvement of poly(ADP-ribosyl)ation in ischemic insult, we examined poly(ADP ribosyl)ation by immunohistochemical methods and the protective effect of 3-aminobenzamide, which is a PARP inhibitor, on focal brain ischemia using an intraluminal permanent middle cerebral artery occlusion model in rats. Poly(ADP ribosyl)ation was widely and markedly detected 2 hours after the ischemic insult in the cerebral cortex and striatum in which infarction developed 24 hours later. The enhanced immunoreactivity of poly(ADP-ribose) gradually decreased, and 16 hours later, no immunoreactivity was detected. Intraventricular administration of 3-aminobenzamide (1 to 30 mg/kg) 30 minutes before the ischemic insult decreased infarction volume in a dose-dependent manner along with the immunohistochemical reduction of poly(ADP-ribosyl)ation. Pretreatment with 7-nitroindazole (25 mg/kg, intraperitoneally), a selective neuronal nitric oxide synthetase inhibitor, partially reduced poly(ADP-ribosyl)ation. These data suggest the involvement of poly(ADP-ribosyl)ation in the development of cerebral infarction.

Animals↗

PubMed1998

Neuroprotective effect of an antioxidant, ebselen, in patients with delayed neurological deficits after aneurysmal subarachnoid hemorrhage.

OBJECTIVE: The effect of ebselen, a seleno-organic compound with antioxidant activity through a glutathione peroxidase-like action, on the outcome of subarachnoid hemorrhage was evaluated in a multicenter placebo-controlled double-blind clinical trial. METHODS: Patients who suffered aneurysmal subarachnoid hemorrhages of Hunt and Kosnik Grades II through IV at admission and were able to start drug treatment within 96 hours of the ictus were enrolled. Early surgery was performed whenever possible. Oral administration of ebselen granules suspended in water (150 mg, twice a day) or placebo was started immediately after admission and continued for 2 weeks. The major end points were the Glasgow Outcome Scale at 2 weeks, 1 month, and 3 months after the start of treatment. The incidence of delayed ischemic neurological deficits clinically diagnosed as resulting from vasospasm and the incidence and extent of low-density areas on postoperative computed tomographic scans were also studied as secondary outcome measures. RESULTS: Intent-to-treat analysis of the 286 patients enrolled in the trial (145 patients administered ebselen and 141 administered placebo) revealed that the incidence of clinically diagnosed delayed ischemic neurological deficits was unaltered. There were 52 (receiving ebselen) and 58 (receiving placebo) patients with delayed deficits; however, a significantly better outcome was observed after ebselen treatment than after placebo (P = 0.005, chi2 test). There was a corresponding decrease in the incidence and extent of low-density areas (P = 0.032, Wilcoxon rank sum test). CONCLUSION: Ebselen reduced brain damage in patients with delayed neurological deficits after subarachnoid hemorrhage and may be a promising neuroprotective agent.

Aged↗

PubMed1998

Consistent injury in the striatum of C57BL/6 mice after transient bilateral common carotid artery occlusion.

OBJECTIVE: The recent availability of transgenic mice enables us to study the functional role of single gene products in cerebral ischemia. To establish an experimental murine model of transient forebrain ischemia, this study examined the temporal profile of ischemic neuronal damage in the striatum after bilateral common carotid artery occlusion. METHODS: C57BL/6 mice, which are frequently used for genetic manipulations, were subjected to 15-minute bilateral common carotid artery occlusion. Ischemic injury was examined (4, 8, 24, 48, and 96 h after reperfusion) by Nissl staining, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end-labeling, and nuclear staining with Hoechst 33258 dye. RESULTS: Regional cerebral blood flow was decreased to 11 +/- 6% of control values during the ischemic insult. Striatal injury was observed in 95% of animals examined after 15-minute bilateral common carotid artery occlusion. The number of small and medium-size neurons in the striatum was significantly (P < 0.05) decreased 8 hours after reperfusion and continued to decrease until 96 hours, whereas the number of large neurons remained constant. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end-labeling-positive cells appeared in the dorsomedial region of the striatum 48 hours after the ischemic insult and throughout the striatum 96 hours after the ischemic insult. Brain sections stained with Hoechst 33258 dye also demonstrated apoptotic nuclei 96 hours after the ischemic insult. CONCLUSION: Striatal injury after transient forebrain ischemia is reproducible in C57BL/6 mice and is a good model to study the molecular mechanisms of ischemic injury, including delayed neuronal death, using transgenic mice.

Animals↗

PubMed1998

The TdT-mediated dUTP nick end labeling assay precisely assesses the DNA damage in human tumor xenografts.

Cultured HL-60, HeLa S3 and WiDr cells grown in male BALB/c nu/nu mice were studied by conventional and field-inversion DNA gel electrophoresis (FIGE), as well as by means of cytomorphological approaches, including TdT-mediated dUTP nick end labeling (TUNEL) assay. Chemosensitivity tests revealed HL-60 to be sensitive to vindesine (VDS), and HeLa S3 and WiDr to mitomycin C (MMC). Although VDS-treated HL-60 exhibited condensation of chromatin and a DNA ladder, MMC-exposed HL-60 cells showed apoptotic figures without typical DNA ladders. With MMC-treated WiDr cells, neither DNA ladders nor apoptotic figures were observed. Cells characterized by chromatin condensation were TUNEL-positive in both treated and untreated cases with the exception of the MMC-treated WiDr case, in which many TUNEL-positive cells were observed without cytomorphological changes. On FIGE, DNA fragments of approximately 50, 300 and 400 kbp were detected in groups treated with both effective and ineffective drugs, as well as in untreated controls. Furthermore, change of the time parameters in FIGE resulted in different sizes (550 and 850 kbp) of DNA fragments. These findings indicate that i) cell death is not always detectable in terms of apoptotic figures or DNA oligonucleosomal fragmentation, ii) only the TUNEL assay is a reliable tool to detect DNA damage and, iii) FIGE does not provide accurate size profiles of macromolecular DNA fragments.

Animals↗

PubMed1998

Do intronic mutations affecting splicing of WT1 exon 9 cause Frasier syndrome?

The WT1 gene, one of the genes responsible for Wilms tumour, is thought to play a crucial role in the development of the kidneys and gonads. This gene encodes four protein isoforms resulting from two alternative splicing sites, one of which involves inclusion or exclusion of lysine, threonine, and serine (KTS) between the third and fourth zinc finger domains. WT1 is virtually always mutationally inactivated in patients with Denys-Drash syndrome. We analysed WT1 in eight patients who had been diagnosed as having this syndrome, and identified five previously unknown mutations affecting splicing donor sites of intron 9. These mutations affect alternative splicing. The isoforms retaining KTS are not produced. The clinical features of the patients with these intronic mutations were consistent with those of Frasier syndrome, characterised by a more slowly progressive nephropathy than Denys-Drash syndrome, associated streak gonads, and no Wilms tumour development. Our results indicate that WT1 isoforms, including/excluding KTS, have different functions in tumorigenesis and organogenesis of the kidneys and gonads.

Adolescent↗