Ikuya Yamaura

Cytotoxic Fragment of Amyloid Precursor Protein Accumulates in Hippocampus After Global Forebrain Ischemia

We developed an antibody specific to β-amyloid precursor protein (βAPP) fragments possessing the exact amino terminus of the β-amyloid peptide and examined its induction in postischemic hippocampus. In control hippocampus, this APP fragment was lightly observed in pyramidal neurons of CA sectors and dentate granule cells. Transient forebrain ischemia enhanced accumulation of the APP fragment in CA1 pyramidal neurons. Seven days after the ischemia, while the APP fragment was still observed in dentate granule cells and CA3 neurons, it disappeared in dead CA1 neurons. While astrocytes did not show in any immunoreactivity throughout the experiment, those in the CA1 sector showed moderate immunoreactivity 7 days after the ischemia. The APP fragment has a cytotoxic effect on cultured neurons. These results suggest that the accumulation of the cytotoxic APP fragment in CA1 neurons may play a role in the development of delayed neuronal death after the ischemic insult.

Calpain inhibitor entrapped in liposome rescues ischemic neuronal damage

Transient forebrain ischemia induces activation of calpain and proteolysis of a neuronal cytoskeleton, fodrin, in gerbil hippocampus. This phenomenon precedes delayed neuronal death in hippocampal CA1 neurons. We examined effects of a calpain inhibitor on delayed neuronal death after transient forebrain ischemia. In gerbils, a selective calpain inhibitor entrapped in liposome was given transvenously and 30 min later, 5-min forebrain ischemia was produced by occlusion of both common carotid arteries. On day 7, CA1 neuronal damage was examined in the hippocampal slices stained with cresyl violet. Calpain-induced proteolysis of fodrin was also examined by immunohistochemistry and immunoblot. Additionally, to assure entrapment of the inhibitor by CA1 neurons, the inhibitor-liposome complex was labeled with FITC and given to gerbils. Fluorescence in the hippocampal slices was examined by confocal laser scanning microscope. Selective CA1 neuronal damage induced by forebrain ischemia was prevented by administration of the inhibitor in a dose-dependent manner. Calpain-induced proteolysis of fodrin was also extinguished by the calpain inhibitor in a dose-dependent manner. Bright fluorescence of the FITC-labeled inhibitor was observed in the CA1 neurons. The data show an important role of calpain in the development of the ischemic delayed neuronal death. Calpain seems to produce neuronal damage by degrading neuronal cytoskeleton. Our data also show a palliative effect of the calpain inhibitor on the neurotoxic damage, which offers a new and potent treatment of transient forebrain cerebral ischemia.

Activation of Protein Kinases in Canine Basilar Artery in Vasospasm

Subarachnoid hemorrhage (SAH) often leads to a long-term narrowing of cerebral artery called vasospasm. To understand the molecular mechanisms in vasospasm, signal transduction of tyrosine kinase pathway and phosphorylation of myosin light chain (MLC) and calponin (CaP) in the basilar artery were studied. Vasospasm was produced in the canine basilar artery by a two-hemorrhage method, and vasocontraction was induced by a local application of KCl or serotonin to the basilar artery after a transclival exposure. Intracellular substrates of tyrosine kinase pathway, including Shc, Raf1, and extracellular-regulated kinases in the basilar artery, were activated after SAH, and the activation of Shc suggests stimulation of signal transductions from tyrosine kinase receptors, G-coupled receptors, or both. The activation of tyrosine kinase pathway in vasospasm also was supported by dose-dependent dilation of the spastic basilar artery on days 0 and 7 by topical application of genistein, a tyrosine kinase inhibitor, and associated marked inhibition of tyrosine phosphorylation of intracellular substrates, including Shc. In addition, the generation of protein kinase M, catalytic fragment of protein kinase Cα (PKCα), in vasospasm on days 0 and 7 was inhibited in response to genistein, indicating an inactivation of μ-calpain. It is suggested, therefore, that the reversal of vasospasm by genistein is closely associated with the restoration of intracellular Ca2+ levels. However, the increased activities of Raf1 and extracellular-regulated kinases in vasospasm were declined on day 7 compared with those on day 0 or 2, suggesting that the activation of tyrosine kinase pathway is more closely associated with the early stage of vasospasm than with the late stage of vasospasm. The analysis by pyrophosphate polyacrylamide gel electrophoresis (PPi-PAGE) demonstrated three MLC bands in vasospasm on days 2 and 7, as well as in KCl- and serotonin-induced vasocontraction. Since PPi-PAGE resolves smooth muscle MLC into three bands in the MLC kinase (MLCK)-mediated phosphorylation and into a single band in the PKC-mediated phosphorylation based on the phosphorylation state, the current results suggest that MLC in vasospasm is phosphorylated by MLCK but not by PKC. In basilar artery, CaP was significantly down-regulated, and in addition, significantly phosphorylated on serine and threonine residues only in vasospasm on days 2 and 7. Although the significance of CaP phosphorylations in vivo still is controversial, CaP down-regulation and phosphorylation may attenuate the inhibition of Mg2+-ATPase activity by CaP and induce a potential enhancement of smooth muscle contractility in delayed vasospasm. Since CaP is phosphorylated vivo by PKC, activated PKC in vasospasm may phosphorylate CaP. Thus, SAH stimulates tyrosine kinase pathway to increase intracellular Ca2+ and activate PKC, and the former activates MLCK to phosphorylate MLC, whereas the latter phosphorylates CaP but not MLC.

Activity of Smooth Muscle Phosphatases 1 and 2A in Rabbit Basilar Artery in Vasospasm

BACKGROUND AND PURPOSE Subarachnoid hemorrhage frequently leads to a long-term cerebral artery narrowing called vasospasm. Recently, the involvement of myosin light chain kinase has been found in experimental vasospasm in our laboratory. We therefore measured the activity of serine/threonine protein phosphatases 1 and 2A in the rabbit basilar artery in vasospasm and in vasocontraction to study their role, particularly in regard to vasospasm compared with vasocontraction. METHODS Vasospasm was produced in the rabbit basilar artery by a two-hemorrhage method. Vasocontraction was induced by local application of KCl or serotonin to the rabbit basilar artery after a transclival exposure. The control animals were treated with saline instead of fresh blood. Serine/threonine protein phosphatase activity in the basilar artery was assayed with the use of [32P]phosphorylase-a as a substrate; protein phosphatase 1 activity was evaluated as protein phosphatase activity in the presence of 1 nmol/L okadaic acid, whereas protein phosphatase 2A activity was assessed as protein phosphatase activity inhibited by 1 nmol/L okadaic acid. RESULTS Values of mean activity of protein phosphatase 1 in myofibrillar extract were 3.58 +/- 0.26 nmol/min per milligram in the control group, 3.22 +/- 0.12 nmol/min per milligram in the spastic group on day 2, and 3.01 +/- 0.16 nmol/min per milligram in the spastic group on day 4 (a significant decrease in protein phosphatase 1 activity in the spastic group on days 2 and 4). In contrast, these values did not show any significant changes in the KCl and serotonin groups. Values of mean activity of protein phosphatase 2A in cytosolic extract were 0.90 +/- 0.07 nmol/min per milligram in the control group, 0.75 +/- 0.10 nmol/min per milligram in the spastic group on day 2, and 0.62 +/- 0.17 nmol/min per milligram in the spastic group on day 4 (a significant reduction in protein phosphatase 2A in the spastic group on days 2 and 4). There was no evidence of significant changes of protein phosphatase 2A in cytosolic extract in the KCl and serotonin groups. CONCLUSIONS Protein phosphatase 1 in myofibrillar extract is reported to catalyze the dephosphorylation of myosin light chain and calponin, whereas protein phosphatase 2A in cytosolic extract catalyzes the dephosphorylation of calponin and caldesmon. In addition, the phosphorylation of calponin and caldesmon results in the loss of their ability to inhibit smooth muscle contraction. Therefore, the significant decrease in activity of protein phosphatases 1 and 2A in vasospasm may result in uninterrupted vascular smooth muscle contraction by the preservation of phosphorylation of not only myosin light chain but also calponin and caldesmon.

Effects of protein kinase C modulators on multidrug resistance in human glioma cells.

To identify the role of protein kinase C (PKC) in multidrug resistance, the effects of phorbol-12-myristate-13-acetate (PMA), a PKC activator, or calphostin C, a PKC inhibitor, on intracellular vincristine accumulation and expression of P-glycoprotein phosphorylation were studied in one multidrug-resistant and three multidrug-sensitive human glioma cell lines. Basal PKC activities and immunoreactivities of PKC-alpha and -zeta were higher in multidrug-resistant cells than in multidrug-sensitive cells. There was no significant difference in the immunoreactivity of PKC-delta between multidrug-resistant and -sensitive cells, and immunoreactive PKC-beta, -gamma, and -epsilon were not detected in either multidrug-resistant or -sensitive cells. The treatment of multidrug-resistant cells with 100 nM PMA for 2 hours resulted in the activation not of PKC-zeta but of PKC-alpha, with concomitant decrease in vincristine accumulation and increase in P-glycoprotein phosphorylation. The exposure of multidrug-resistant cells to 100 nM PMA for 24 hours induced down-regulation not of PKC-zeta but of PKC-alpha, with concurrent decrease in vincristine accumulation, and reduced but still increased P-glycoprotein phosphorylation. The treatment of multidrug-resistant cells with 100 nM calphostin C for 2 hours decreased immunoreactive PKC-zeta and not immunoreactive PKC-alpha, inducing increase in vincristine accumulation, with concomitant decrease in P-glycoprotein phosphorylation. There was no evidence of significant change in vincristine accumulation in multidrug-sensitive cells treated with PMA or calphostin C. This may suggest that at least two isozymes of PKC, PKC-alpha and -zeta, are involved in P-glycoprotein phosphorylation and that vincristine efflux function in multidrug-resistant human glioma cells is closely associated with P-glycoprotein phosphorylation and is decreased by PKC inhibitor.

Spontaneous spinal epidural hematoma with hemiparesis mimicking acute cerebral infarction: Two case reports

Abstract Context Acute hemiparesis is a common initial presentation of ischemic stroke. Although hemiparesis due to spontaneous spinal epidural hematoma (SSEH) is an uncommon symptom, a few cases have been reported and misdiagnosed as cerebral infarction. Design Case reports of SSEH with acute hemiparesis. Findings In these two cases, acute stroke was suspected initially and administration of intravenous alteplase therapy was considered. In one case, the presentation was neck pain and in the other case, it was Lhermittes sign; brain magnetic resonance imaging (MRI) and magnetic resonance angiography were negative for signs of ischemic infarction, hemorrhage, or arterial dissection. Cervical MRI was performed and demonstrated SSEH. Conclusion Clinicians who perform intravenous thrombolytic treatment with alteplase need to be aware of this possible contraindication.

Immunohistochemistry of leukotriene C4 in experimental cerebral vasospasm.

SummaryExperimental cerebral vasospasm was produced in a “two-hemorrhage” canine model and examined by immunohistochemistry for leukotriene C4 (LTC4). The immunostain for LTC4 showed a strong positivity in intima and adventitia and a scattered reaction in media of normal basilar artery. The immunoreactivity after subarachnoid hemorrhage (SAH) was little changed in intima and media. Inflammatory cells which were characterized histochemically as neutrophils and macrophages, were shown to infiltrate from the adventitia of basilar artery to the periphery of blood clot after SAH and were markedly immunoreactive for LTC4. Also the neutrophils increased in number with the lapse of time after SAH. Thus, it would be reasonable to conclude that the LTC4 responsible for the development of vasospasm would most likely be produced from the infiltrating neutrophils and macrophages. In addition, neurons in hypothalamus, median eminence, and pons, as well as ependymal and arachnoid cells were immunoreactive for LTC4 both in the control and after SAH, whereas astrocytes and oligodendrocytes were not immunoreactive for LTC4 in either case.

Immunoblotting of Contractile and Cytoskeletal Proteins of Canine Basilar Artery in Vasospasm

Vasospasm was produced in the canine basilar arteries by a two-hemorrhage method, and voltage- and receptor-dependent contractions of the normal canine basilar arteries were induced by local applications of potassium chloride (KCI) and serotonin, respectively, after transclival exposure. Actin, myosin, desmin, filamin, talin, vinculin, and alpha-actinin in the basilar artery were studied by immunoblotting. The immunoblots showed a decrease or loss in immunoreactivity of some native proteins and generation of protein fragments, smaller in size than native proteins, in spastic, KCI, and serotonin groups, indicating a proteolytic degradation. In the spastic group on Day 2, actin, desmin, and filamin were usually degraded slightly; myosin moderately; and talin and alpha-actinin substantially. Vinculin and metavinculin remained intact. In the spastic group on Day 7, actin and desmin were usually decomposed slightly; myosin, filamin, and vinculin substantially; and talin, metavinculin, and alpha-actinin markedly. In the KCI and serotonin groups, slight degradation was usually observed in filamin, often in alpha-actinin, and occasionally in actin, whereas desmin, vinculin, and metavinculin were not degraded. In addition, myosin was usually degraded moderately in the KCI group and slightly in the serotonin group, and talin was generally decomposed slightly in the KCI group and moderately in the serotonin group. The degraded fragments, although variable in number and immunoreactivity, were similar in size in the three groups. We suggest that the intracellular devices responsible for contraction of the basilar arteries are degraded more severely in the spastic group than in the KCI or serotonin group, probably by similar proteolytic mechanism and progressively with the passage of time after subarachnoid hemorrhage in vasospasm.

The immunohistochemical distribution of protein kinase C isozymes is altered in the canine brain and basilar artery after subarachnoid hemorrhage

The effects of subarachnoid hemorrhage on immunoreactivities of protein kinase C isozymes (alpha, beta, gamma) were studied in canine brain and basilar artery. Mild and severe constriction of the basilar artery was shown 2 days and 7 days after hemorrhage, respectively. In control brain tissues, the isozymes showed distinct distributions and following hemorrhage, reactive astrocytes with protein kinase C alpha staining emerged in the surface of the pons and hippocampus. The control basilar artery showed protein kinase C alpha staining and its staining was decreased on day 7, consistent with the result of immunoblot. Our results demonstrated that subarachnoid hemorrhage induces gliosis with heavy protein kinase C alpha staining and down-regulation of protein kinase C in the artery.

Meningioma in Sigmoid Sinus Groove Associated with Dural Arteriovenous Malformation

A 65-year-old woman was evaluated for vertigo. Computed tomography and magnetic resonance imaging as well as angiograghy revealed the coexistence of a tumor at the junction of the right transverse-sigmoid sinuses, a dural arteriovenous malformation (AVM) of the right transverse sinus, and a right sigmoid sinus thrombosis. After transarterial embolization via the external carotid artery, the tumor was totally removed and the right transverse sinus was embolized with platinum coils and Surgicel. Postoperatively, the dural AVM was completely occluded, and the histological examination of the tumor showed it to be a transitional type of meningioma