Chisato Kubota

Constitutive Reactive Oxygen Species Generation from Autophagosome/Lysosome in Neuronal Oxidative Toxicity

Reactive oxygen species (ROS) are involved in several cell death processes, including cerebral ischemic injury. We found that glutamate-induced ROS accumulation and the associated cell death in mouse hippocampal cell lines were delayed by pharmacological inhibition of autophagy or lysosomal activity. Glutamate, however, did not stimulate autophagy, which was assessed by a protein marker, LC3, and neither changes in organization of mitochondria nor lysosomal membrane permeabilization were observed. Fluorescent analyses by a redox probe PF-H2TMRos revealed that autophagosomes and/or lysosomes are the major sites for basal ROS generation in addition to mitochondria. Treatments with inhibitors for autophagy and lysosomes decreased their basal ROS production and caused a burst of mitochondrial ROS to be delayed. On the other hand, attenuation of mitochondrial activity by serum depletion or by high cell density culture resulted in the loss of both constitutive ROS production and an ROS burst in mitochondria. Thus, constitutive ROS production within mitochondria and lysosomes enables cells to be susceptible to glutamate-induced oxidative cytotoxicity. Likewise, inhibitors for autophagy and lysosomes reduced neural cell death in an ischemia model in rats. We suggest that cell injury during periods of ischemia is regulated by ROS-generating activity in autophagosomes and/or lysosomes as well as in mitochondria.

Experimental Model of Lacunar Infarction in the Gyrencephalic Brain of the Miniature Pig. Neurological Assessment and Histological, Immunohistochemical, and Physiological Evaluation of Dynamic Corticospinal Tract Deformation

Background and Purpose— Lacunar infarction accounts for 25% of ischemic strokes, but the pathological characteristics have not been investigated systematically. A new experimental model of lacunar infarction in the miniature pig was developed to investigate the pathophysiological changes in the corticospinal tract from the acute to chronic phases. Methods— Thirty-five miniature pigs underwent transcranial surgery for permanent anterior choroidal artery occlusion. Animals recovered for 24 hours (n=7), 2 (n=5), 3 (n=2), 4 (n=2), 6 (n=1), 7 (n=7), 8 (n=2), and 9 days (n=1), 2 weeks (n=2), 4 weeks (n=3), and more than 4 weeks (n=3). Neurology, electrophysiology, histology, and MRI were performed. Seven additional miniature pigs underwent transient anterior choroidal artery occlusion to study muscle motor-evoked potentials and evaluate corticospinal tract function during transient anterior choroidal artery occlusion. Results— The protocol had a 91.4% success rate in induction of internal capsule infarction 286±153 mm3 (mean±SD). Motor-evoked potentials revealed the presence of penumbral tissue in the internal capsule after 6 to 15 minutes anterior choroidal artery occlusion. Total neurological deficit scores of 15.0 (95% CI, 13.5 to 16.4) and 3.4 (0.3 to 6.4) were recorded for permanent anterior choroidal artery occlusion and sham groups, respectively (P<0.001, maximum score 25) with motor deficit scores of 3.4 (95% CI, 2.9 to 4.0) and 0.0 (CI, 0.0 to 0.0), respectively (P<0.001, maximum score 9). Histology revealed that the internal capsule lesion expands gradually from acute to chronic phases. Conclusions— This new model of lacunar infarction induces a reproducible infarct in subcortical white matter with a measurable functional deficit and evidence of penumbral tissue acutely.

Cilostazol Attenuates Gray and White Matter Damage in a Rodent Model of Focal Cerebral Ischemia

Background and Purpose— To evaluate whether delayed treatment with the antiplatelet agent cilostazol reduces the volume of infarction in the gray and white matter in a rodent model of permanent focal cerebral ischemia and to explore the mechanism of the neuroprotective effect in vivo. Methods— Cilostazol (30 or 50 mg/kg) or vehicle was administered by gavage 30 minutes and 4 hours after the induction of cerebral ischemia by permanent occlusion of the left middle cerebral artery (MCA). Animals were euthanized 24 hours after MCA occlusion, and the volume of gray matter damage was evaluated by quantitative histopathology. Axonal damage was determined with amyloid precursor protein immunohistochemistry. Dynamic susceptibility contrast MRI was used to assess regional cerebral blood volume (CBV) and cerebral blood flow (CBF). Results— Treatment with the higher dose of cilostazol (50 mg/kg) significantly reduced the volume of gray matter damage and axonal damage in the cerebral hemisphere by 45.0% (P<0.02) and 42.4% (P<0.002), respectively, compared with the control group. Relative CBV in the peri-infarct area after MCA occlusion was significantly increased in the cilostazol-treated group (50 mg/kg) compared with the control group (P<0.05). Relative CBF tended to be higher in the cilostazol-treated group compared with the control group. Conclusions— Treatment with cilostazol significantly reduced the gray and white matter damage associated with permanent focal ischemia. Cilostazol improved CBV and CBF in the peri-infarct area. The major action of cilostazol is to increase perfusion in the ischemic penumbra.

Experimental investigation of encephalomyosynangiosis using gyrencephalic brain of the miniature pig: histopathological evaluation of dynamic reconstruction of vessels for functional anastomosis. Laboratory investigation.

OBJECT Encephalomyosynangiosis (EMS) is a surgical treatment for moyamoya disease that is widely used to provide increased intracranial blood flow via revascularization by arterial anastomosis from the external carotid artery. However, the angiogenic mechanism responsible for the revascularization induced by EMS has not been systematically evaluated. In this study the authors investigated the chronological angiogenic changes associated with EMS to clarify the favorable factors and identify revascularization mechanisms by using an experimental internal carotid artery occlusion (ICAO) model in the miniature pig. METHODS Fourteen miniature pigs were used, 11 of which underwent ICAO before transcranial surgery for EMS was performed. Animals were allowed to recover for 1 week (4 pigs) or 4 weeks (7 pigs) after EMS. Control group animals were treated in the same way, but without occlusion (3 pigs). Magnetic resonance imaging, angiography, and histological investigation were performed. RESULTS One week after EMS, on histological examination of both the ICAO and control groups it was found that the transplanted temporal muscle had adhered to the arachnoid via a granulation zone, which was enriched with immune cells such as macrophages associated with the angiogenic process. Four weeks after EMS, angiography and histological examination of the ICAO group showed patent anastomoses between the external carotid artery and the cortical arteries without any detectable boundary between the temporal muscle and the cerebral cortex. In contrast, histological examination of the control group found scar tissue between the cerebral cortex and temporal muscle. CONCLUSIONS The initial step for formation of anastomoses resembles the process of wound healing associated with repair processes such as active proliferation of macrophages and angiogenesis within the new connective tissue. Functional revascularization requires a suitable environment (such as tissue containing vascular beds) and stimulus (such as ischemia) to induce vascular expansion.

Lipoxygenase-mediated generation of lipid peroxides enhances ferroptosis induced by erastin and RSL3

In cancer cells the small compounds erastin and RSL3 promote a novel type of cell death called ferroptosis, which requires iron‐dependent accumulation of lipid reactive oxygen species. Here we assessed the contribution of lipid peroxidation activity of lipoxygenases (LOX) to ferroptosis in oncogenic Ras‐expressing cancer cells. Several 12/15‐LOX inhibitors prevented cell death induced by erastin and RSL3. Furthermore, siRNA‐mediated silencing of ALOX15 significantly decreased both erastin‐induced and RSL3‐induced ferroptotic cell death, whereas exogenous overexpression of ALOX15 enhanced the effect of these compounds. Immunofluorescence analyses revealed that the ALOX15 protein consistently localizes to cell membrane during the course of ferroptosis. Importantly, treatments of cells with ALOX15‐activating compounds accelerated cell death at low, but not high doses of erastin and RSL3. These observations suggest that tumor ferroptosis is promoted by LOX‐catalyzed lipid hydroperoxide generation in cellular membranes.

The pseudophosphatase phogrin enables glucose-stimulated insulin signaling in pancreatic β-cells

Autocrine insulin signaling is critical for pancreatic β-cell growth and activity and is at least partially controlled by protein-tyrosine phosphatases (PTPs) that act on insulin receptors (IRs). The receptor-type PTP phogrin primarily localizes on insulin secretory granules in pancreatic β cells. We recently reported that phogrin knockdown decreases the protein levels of insulin receptor substrate 2 (IRS2), whereas high-glucose stimulation promotes formation of a phogrin–IR complex that stabilizes IRS2. However, the underlying molecular mechanisms by which phogrin affects IRS2 levels are unclear. Here, we found that relative to wildtype mice, IRS2 levels in phogrin-knockout mice islets decreased by 44%. When phogrin was silenced by shRNA in pancreatic β-cell lines, glucose-induced insulin signaling led to proteasomal degradation of IRS2 via a negative feedback mechanism. Phogrin overexpression in a murine hepatocyte cell line consistently prevented chronic insulin treatment–induced IRS2 degradation. In vitro, phogrin directly bound the IR without the assistance of other proteins and protected recombinant PTP1B from oxidation to potentiate its activity toward the IR. Furthermore, phogrin expression suppressed insulin-induced local generation of hydrogen peroxide and subsequent PTP1B oxidation, which allowed progression of IR dephosphorylation. Together, these results suggest that a transient interaction of phogrin with the IR enables glucose-stimulated autocrine insulin signaling through the regulation of PTP1B activity, which is essential for suppressing feedback-mediated IRS2 degradation in pancreatic β cells.

Impaired Processing of Prohormones in Secretogranin III–Null Mice Causes Maladaptation to an Inadequate Diet and Stress

Secretogranin III (SgIII), a member of the granin family, binds both to another granin, chromogranin A (CgA), and to a cholesterol-rich membrane that is destined for secretory granules (SGs). The knockdown of SgIII in adrenocorticotropic hormone (ACTH)-producing AtT-20 cells largely impairs the regulated secretion of CgA and ACTH. To clarify the physiological roles of SgIII in vivo, we analyzed hormone secretion and SG biogenesis in newly established SgIII-knockout (KO) mice. Although the SgIII-KO mice were viable and fertile and exhibited no overt abnormalities under ordinary rearing conditions, a high-fat/high-sucrose diet caused pronounced obesity in the mice. Furthermore, in the SgIII-KO mice compared with wild-type (WT) mice, the stimulated secretion of active insulin decreased substantially, whereas the storage of proinsulin increased in the islets. The plasma ACTH was also less elevated in the SgIII-KO mice than in the WT mice after chronic restraint stress, whereas the storage level of the precursor proopiomelanocortin in the pituitary gland was somewhat increased. These findings suggest that the lack of SgIII causes maladaptation of endocrine cells to an inadequate diet and stress by impairing the proteolytic conversion of prohormones in SGs, whereas SG biogenesis and the basal secretion of peptide hormones under ordinary conditions are ensured by the compensatory upregulation of other residual granins or factors.

A new model of focal cerebral ischemia in the miniature pig

Object The purpose of this set of studies is to design a minimally invasive, reproducible stroke model in the gyrencephalic brain. This paper provides information on both surgical technique and methods of quantification of ischemic damage to both gray and white matter in the miniature pig. Methods Sixteen male miniature pigs were randomly divided into three groups and underwent transcranial surgery involving a frontotemporal approach with orbital rim osteotomy for permanent middle cerebral artery occlusion (MCAO; five animals), permanent internal carotid artery occlusion (ICAO; six animals), and a sham operation (five animals). Histological mapping and magnetic resonance (MR) imaging were used to delineate the areas of ischemic damage. The volumes of infarction measured directly from MR images were 16.2 ± 1.1, 1.5 ± 0.5, and 0.0 ± 0.0 cm3 (mean ± standard deviation [SD], p < 0.001) in the MCAO, ICAO, and sham-operated groups, respectively. The areas of ischemia identified through histological analysis and...