Sadaharu Tabuchi

Expression of lysophosphatidic acid receptor in rat astrocytes: mitogenic effect and expression of neurotrophic genes.

Lysophosphatidic acid (LPA) is a phospholipid mediator with a variety of biological activities. It remains unknown, however, which cells in the brain express the LPA receptor. The present study was undertaken to identify cells in the rat brain expressing functional LPA receptors, and to explore biological roles of LPA in these cells. We found that the LPA receptor was most dominantly expressed in rat astrocytes, determined by LPA-induced Ca2+ imaging, and by Northern blot analyses. LPA induced a mitogenic response and expression of immediate early genes in astrocytes, through pertussis-toxin sensitive G-protein(s). LPA also stimulated the expression of various cytokine genes, including nerve growth factor, interleukin (IL)-1β, IL-3 and IL-6. Thus, astrocytes are the major target of LPA in the brain. We propose that LPA may play important roles in neuronal development, gliosis and wound-healing process in the brain.

Mice deficient in cytosolic phospholipase A2 are less susceptible to cerebral ischemia/reperfusion injury

To determine the role of cytosolic phospholipase A2 (cPLA2) in infarct development, wild-type and cPLA2 knock-out mice were subjected to focal cerebral ischemia for 75 min by occluding the middle cerebral artery using nylon filament and subsequent reperfusion by withdrawing the filament. The neurological deficit severity was evaluated by a modified 4-point scale. After the reperfusion period (72 h), mice were killed, and the brains were cut into four 2 mm coronal sections using a rodent brain matrix. Sections were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC). The infarct volume was 87.19 +/- 27.54 mm3 (mean +/- SD, n = 11) in the wild-type mice and 48.20 +/- 31.32 mm3 (n = 10; P < 0.01 vs. wild-type) in the knock-out mice. Less severe functional neurological deficits were observed in knock-out mice at 72 h after ischemia when compared with wild-type. Thus, disruption of cPLA2 resulted in significant reduction of infarct area and neurological deficit severity in the MCA occlusion model. These data indicate a critical role for cPLA2 in the pathogenesis of cerebral ischemia/ reperfusion injury.

Nonenzymatic derived lipid peroxide, 8-iso-PGF2α, participates in the pathogenesis of delayed cerebral vasospasm in a canine SAH model

Abstract We studied whether 8-iso-PGF2α, nonenzymatic arachidonyl peroxide, participated in the pathogenesis of delayed vasospasm using a canine subarachnoid hemorrhage (SAH) model. Fourteen adult mongrel dogs were divided into two groups, two-hemorrhage SAH group (n = 8) and control group (n = 6). The contents of 8-iso-PGF 2α in CSF, the basilar artery segment, and subarachnoid clot were measured by enzyme immunoassay kit. The CSF 8-iso-PGF2α content on Day 7 in the SAH group was 67.9 ± 29.9 pg ml-1 (n = 8), which was significantly higher than 27.1 ± 13.8 (n = 8) on Day 0 in the SAH group, and 33.2 ± 14.4 pg ml-1 (n = 5) on Day 7 in the control group. The 8-iso-PGF2α content in the basilar artery segment with spasm on Day 7 in the SAH group was 13.5 ± 1.9 pg mg-1 wet weight (n = 8), significantly higher than 8.7 ± 1.9 (n = 6) in the control group. The 8-iso-PGF2α content in subarachnoid clot was 1.7 ± 1.4 ng g-1 wet weight (n = 8). Significant elevation of the 8-iso-PGF2α contents in the CSF and the basilar artery segment occurred on Day 7 in the SAH group. The subarachnoid clot enclosed the basilar artery on Day 7, contained a considerable amount of 8-iso-PGF2α. These results suggested that 8-iso-PGF2α could play a crucial role in the pathogenesis of the delayed cerebral vasospasm. [Neurol Res 2002; 24: 301-306]

Calpain induces proteolysis of neuronal cytoskeleton in ischemic gerbil forebrain

We investigated the relationship between the activity of calcium-dependent protease (calpain) and the ischemic neuronal damage. We also investigated the mechanism of ischemic resistance in astrocytes. In gerbil, a 10-min forebrain ischemia was induced by occlusion of both common carotid arteries. The calpain-induced proteolysis of cytoskeleton (fodrin) was examined by immunohistochemistry. Immunolocalization of micro and m-calpain was also examined. Intact fodrin was observed both in neurons and astrocytes, but proteolyzed fodrin was not observed in normal brain. Fifteen minutes after ischemia, proteolysis of fodrin took place in putamen, parietal cortex and hippocampal CA1. The proteolysis extended to thalamus 4 h after ischemia after which the immunoreactivity faded down in all areas except hippocampus. On day 7, the proteolysis was still observed only in hippocampus. Neurons with the proteolysis of soma resulted in neuronal death. Throughout the experiment, the proteolysis was not observed in astrocytes. micro -Calpain was observed only in neurons but m-calpain was observed both in neurons and astrocytes. The ischemia induced only micro -calpain activation, which resulted in fodrin proteolysis of neurons with differential spatial distribution and temporal course. The proteolysis was developed rapidly and was completed within 24 h in all vulnerable regions except hippocampal CA1. The proteolysis preceded the neuronal death. The mechanism of the proteolysis seemed to be involved by Ca(2+) influx via glutamate receptor and rapid neuronal death seemed reasonable. The reason why neuronal death in CA1 evolved slowly was not clarified. In astrocytes, fodrin was not proteolyzed by m-calpain. The low Ca(2+)-sensitivity of m-calpain may be the reason of ischemic resistance in astrocytes.

Lipid mediators modulate NMDA receptor currents in a Xenopus oocyte expression system.

We determined the modulatory effects of various lipid mediators on mouse N-methyl-D-aspartate (NMDA) receptor currents in the Xenopus oocyte expression system. Arachidonic acid, but not oleic acid potentiated NMDA receptor activity. The epsilon1/zeta1 heterodimer of the NMDA receptor was more sensitive to arachidonic acid than was the epsilon2/zeta1 heterodimer. Platelet-activating factor (PAF) and lysophosphatidic acid (LPA) both activated the NMDA currents, and the effects were more evident in the epsilon2/zeta1 heterodimer than in epsilon1/zeta1. These activations were abolished by treatment with protein kinase inhibitors, suggesting a possible phosphorylation of the receptor. Thus, lipid mediators do have modulatory effects on NMDA receptor currents, the potentiating effects of which differ depending on subtype of the NMDA receptor.

A non-enzymatic derived arachidonyl peroxide, 8-iso-prostaglandin F2α, in cerebrospinal fluid of patients with aneurysmal subarachnoid hemorrhage participates in the pathogenesis of delayed cerebral vasospasm

We performed serial measurements of 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), a non-enzymatic derived arachidonyl peroxide, in the cerebrospinal fluid (CSF) of 34 patients with subarachnoid hemorrhage (SAH). Patients were treated with open or endovascular surgery within 48 h of onset. Delayed cerebral vasospasm was verified by the presence of a low-density area on CT scan indicating focal cerebral infarction occurring after symptomatic delayed vasospasm. Concentrations of 8-iso-PGF2alpha in the CSF of 15 patients exhibiting delayed cerebral vasospasm were compared with those of 19 patients who did not exhibit vasospasm. The concentrations of 8-iso-PGF2alpha in the CSF of patients showing vasospasm were 42.4+/-37.1 pg/ml (mean+/-S.D., n=12) on Days 0-2, 66.4+/-41.0 pg/ml (n=14) on Days 3-5, 118.5+/-89.9 pg/ml (n=15) on Days 6-8, 86.2+/-70.2 pg/ml (n=11) on Days 9-11, 48.8+/-31.8 pg/ml (n=10) on Days 12-14, 27.8+/-20.1 pg/ml (n=7) after Day 20, while the concentrations in patients not showing vasospasm were 24.8+/-12.0 pg/ml (n=18) on Days 0-2, 25.7+/-15.2 pg/ml (n=19) on Days 3-5, 47.5+/-52.3 pg/ml (n=18) on Days 6-8, 56.7+/-72.0 pg/ml (n=13) on Days 9-11, 34.2+/-53.1 pg/ml (n=15) on Days 12-14, 20.1+/-18.2 pg/ml (n=10) after Day 20. CSF concentrations of 8-iso-PGF2alpha on Days 3-5 and Days 6-8 were significantly higher in patients showing vasospasm as compared to patients not showing vasospasm. CSF levels of 8-iso-PGF2alpha in patients showing vasospasm gradually increased in the days after onset of SAH and peaked on Days 6-8. Levels returned to normal after Day 20. These values on Days 3-5, Days 6-8, and Days 9-11 were significantly higher than the value after Day 20. Considering these data and the biological activities of 8-iso-PGF2alpha, such as development of inflammation, membrane perturbation and vasoconstriction, we conclude that 8-iso-PGF2alpha may play a role in delayed cerebral vasospasm after SAH.

Spatial resolution of calpain-catalyzed proteolysis in focal cerebral ischemia.

Transient forebrain ischemia induces calpain-mediated degradation of the neuronal cytoskeleton, alpha-fodrin, and this results in ischemic neuronal death. In this study, we investigated the spatial distribution and temporal changes of calpain-catalyzed alpha-fodrin proteolysis in focal cerebral ischemia and examined the effects of a calpain inhibitor. Ischemia was induced in gerbils by 3-h middle cerebral artery occlusion followed by reperfusion. Animals were divided into four groups: a sham-operated group, an ischemic group, a vehicle-treated group, and a calpain inhibitor-treated group. Intravenous injections of vehicle or calpain inhibitor I were administered 30 min before ischemia. Infarct volumes were measured 1 day after reperfusion and the spatial distribution of calpain-catalyzed alpha-fodrin proteolysis was investigated by immunohistochemistry 15 min, 1 h, 4 h, and 1 day after reperfusion. Infarct volume (mean +/- SD) in the ischemic group and the vehicle-treated group was 204.6 +/- 19.1 mm3 and 212.4 +/- 16.3 mm3, respectively, and the calpain inhibitor I reduced the infarct volume [149.4 +/- 25.2 mm3 (P < 0.05)]. Immunoblot analysis demonstrated that calpain inhibitor reduced proteolysis. Ischemia induced fodrin proteolysis in the ischemic core and the peri-infarct zone within 15 min after reperfusion, with proteolysis developing quickly in the ischemic core and more slowly in the peri-infarct zone. Proteolysis preceded neuronal death in the peri-infarct zone. Calpain inhibitor I ameliorated neuronal death in the peri-infarct zone but not in the ischemic core. Thus, calpain plays a pivotal role on focal ischemia as well as in global ischemia.

Platelet-activating factor and somatostatin activate mitogen-activated protein kinase (MAP kinase) and arachidonate release

Platelet-activating factor (PAF) receptor and somatostatin receptor (SSTR4) were cloned, and their primary structures were identified. They are both highly expressed in the rat hippocampus. When expressed in Chinese hamster ovary cells, these receptors activated mitogen-activated protein (MAP) kinase cascade and phospholipase A2. Arachidonic acid or its derivatives, thus produced by the activation of these receptors may play some roles in synaptic transmission and synaptic plasticity.

Neuroendoscopic surgery for ventriculitis and hydrocephalus after shunt infection and malfunction: Preliminary report of a new strategy.

If not controlled in the early stage, ventriculitis is difficult to treat neurosurgically and can lead to serious sequelae, a long course of treatment, and hospitalization. We report two cases of ventriculitis and progressive hydrocephalus after shunt infection. Both were successfully treated by neuroendoscopic septostomy in combination with thorough intraventricular irrigation through a single burr hole followed by single shunt revision. Although surgical intervention has not been established as a first‐choice treatment for ventriculitis, including early‐stage ventriculitis, prompt neuroendoscopic surgery appears effective for the management of ventriculitis and hydrocephalus after shunt infection. The strategy described in this report might be useful to avoid recurrent shunt infections and malfunctions, simplify a shunt, and reduce the overall duration of hospitalization.

Relationship between Postmenopausal Estrogen Deficiency and Aneurysmal Subarachnoid Hemorrhage

Aneurysmal subarachnoid hemorrhage (SAH) is one of the most severe forms of stroke, which results from the rupture of a cerebral aneurysm. SAH is the only type of stroke with a female predominance, suggesting that reproductive factors may play a significant role in the etiology. Estrogen has important effects on vascular physiology and pathophysiology of cerebral aneurysm and SAH and, thus, potential therapeutic implications. There have been growing bodies of epidemiological and experimental studies which support the hypothesis of a significant relationship between estrogen deficiency and cerebral aneurysm formation with subsequent SAH. This hypothesis is the focus of this review as well as possible pathology-based therapeutics with regard to aspects of molecular pathophysiology, especially related to womens health.