Yasuo Aihara

Molecular cloning of a novel brain-type Na(+)-dependent inorganic phosphate cotransporter.

Abstract: We have isolated a human cDNA encoding a protein, designated DNPI, that shows 82% amino acid identity and 92% similarity to the human brain‐specific Na+‐dependent inorganic phosphate (Na+/Pi) cotransporter (BNPI), which is localized exclusively to neuron‐rich regions. Expression of DNPI mRNA in Xenopus oocytes resulted in a significant increase in Na+‐dependent Pi transport, indicating that DNPI is a novel Na+/Pi cotransporter. Northern blot analysis shows that DNPI mRNA is expressed predominantly in brain, where the highest levels are observed in medulla, substantia nigra, subthalamic nucleus, and thalamus, all of which express BNPI mRNA at low levels. In contrast, DNPI mRNA is expressed at low levels in cerebellum and hippocampus, where BNPI mRNA is expressed at high levels. No hybridizing signal for DNPI mRNA is observed in the glia‐rich region of corpus callosum. In other regions examined, both mRNAs are moderately or highly expressed. These results indicate that BNPI and DNPI, which coordinate Na+‐dependent Pi transport in the neuron‐rich regions of the brain, may form a new class within the Na+/Pi cotransporter family.

Quantitative Analysis of Gene Expressions Related to Inflammation in Canine Spastic Artery After Subarachnoid Hemorrhage

Background and Purpose— The possible role of inflammatory reaction of the cerebral artery in the pathogenesis of cerebral vasospasm has been noted in recent studies. We quantitatively measured the levels of expression of genes related to inflammation in the spastic artery in a canine double-hemorrhage model. Methods— Twenty dogs were assigned to 4 groups: group D0, control; group D2, dogs killed 2 days after cisternal injection of blood; group D7, dogs given double cisternal injections of blood and killed 7 days after the first injection; and group D14. Angiography was performed twice: on the first day and before the animals were killed. Total RNA was extracted from the basilar artery. The expressions of interleukin (IL)-1&agr;, IL-6, IL-8, IL-10, tumor necrosis factor-&agr;, E-secretin, fibronectin, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule-1, transforming growth factor-&bgr;, basic fibroblast growth factor, and collagen types I, III, and IV were examined with TaqMan real-time quantitative reverse transcription–polymerase chain reaction. Results— Prolonged arterial narrowing peaking on 7 day was observed. There was a significant difference in vessel caliber between D0, D2, D7, and D14 groups (P <0.0001). There were significant differences in mRNA expression in the basilar artery for IL-1&agr;, IL-6, IL-8, ICAM-1, and collagen type I between D0, D2, D7, and D14 groups (P =0.0079, 0.0196, 0.0040, 0.0017, and <0.0001, respectively). The average level of mRNA was highest in D7 for IL-1&agr;, IL-6, IL-8, and ICAM-1 (17-, 16-, 131-, and 1.7-fold compared with those of D0, respectively) and in D14 for collagen type I (10.9-fold). Conclusions— Increased expression of genes related to inflammation in the spastic artery suggests that inflammatory reaction of the cerebral artery is associated with sustained contraction.

Novel mechanism of endothelin-1-induced vasospasm after subarachnoid hemorrhage

Cerebral vasospasm is a major cause of morbidity and mortality after aneurysmal subarachnoid hemorrhage (SAH). It is a sustained constriction of the cerebral arteries that can be reduced by endothelin (ET) receptor antagonists. Voltage-gated Ca2+ channel antagonists such as nimodipine are relatively less effective. Endothelin-1 is not increased enough after SAH to directly cause the constriction, so we sought alternate mechanisms by which ET-1 might mediate vasospasm. Vasospasm was created in dogs, and the smooth muscle cells were studied molecularly, electro-physiologically, and by isometric tension. During vasospasm, ET-1, 10 nmol/L, induced a nonselective cation current carried by Ca2+ in 64% of cells compared with in only 7% of control cells. Nimodipine and 2-aminoethoxydiphenylborate (a specific antagonist of store-operated channels) had no effect, whereas SKF96365 (a nonspecific antagonist of nonselective cation channels) decreased this current in vasospastic smooth muscle cells. Transient receptor potential (TRP) proteins may mediate entry of Ca2+ through nonselective cationic pathways. We tested their role by incubating smooth muscle cells with anti-TRPC1 or TRPC4, both of which blocked ET-1-induced currents in SAH cells. Anti-TRPC5 had no effect. Anti-TRPC1 also inhibited ET-1 contraction of SAH arteries in vitro. Quantitative polymerase chain reaction and Western blotting of seven TRPC isoforms found increased expression of TRPC4 and a novel splice variant of TRPC1 and increased protein expression of TRPC4 and TRPC1. Taken together, the results support a novel mechanism whereby ET-1 significantly increases Ca2+ influx mediated by TRPC1 and TRPC4 or their heteromers in smooth muscle cells, which promotes development of vasospasm after SAH.

Molecular Profile of Vascular Ion Channels After Experimental Subarachnoid Hemorrhage

Cerebral vasospasm is a transient, delayed constriction of cerebral arteries that occurs after subarachnoid hemorrhage (SAH). Smooth muscle cells show impaired relaxation after SAH, which may be caused by a defect in the ionic mechanisms regulating smooth muscle membrane potential and Ca2+ permeability. We tested this hypothesis by examining changes in expression of mRNA and protein for ion channels in the basilar arteries of dogs after SAH using quantitative real-time polymerase chain reaction (PCR) and western blotting. SAH was associated with a significant reduction in basilar artery diameter to 41 ± 8% of pre-SAH diameter (P < 0.001) after 7 days. There was significant downregulation of the voltage-gated K+ channel Kv 2.2 (65% reduction in mRNA, P < 0.001; 49% reduction in protein, P < 0.05) and the β1 subunit of the large-conductance, Ca2+-activated K+ (BK) channel (53% reduction in mRNA, P < 0.02). There was no change in BK β1 subunit protein. Changes in mRNA levels of Kv 2.2 and the BK-β1 subunit correlated with the degree of vasospasm (r2 = 0.490 and 0.529 respectively, P < 0.05). The inwardly rectifying K+ (Kir) channel Kir 2.1 was upregulated (234% increase in mRNA, P < 0.001; 350% increase in protein, P < 0.001). There was no significant change in mRNA expression of L- type Ca2+ channels and the BK-α subunit. These data suggest that K+ channel dysfunction may contribute to the pathogenesis of cerebral vasospasm.

Voltage-gated K + channel dysfunction in myocytes from a dog model of subarachnoid hemorrhage

Delayed cerebral vasospasm after subarachnoid hemorrhage is primarily due to sustained contraction of arterial smooth muscle cells. Its pathogenesis remains unclear. The degree of arterial constriction is regulated by membrane potential that in turn is determined predominately by K+ conductance (GK). Here, we identified the main voltage-gated K+ (Kv) channels contributing to outward delayed rectifier currents in dog basilar artery smooth muscle as Kv2 class through a combination of electrophysiological and pharmacological methods. Kv2 current density was nearly halved in vasospastic myocytes after subarachnoid hemorrhage (SAH) in dogs, and Kv2.1 and Kv2.2 were downregulated in vasospastic myocytes when examined by quantitative mRNA, Western blotting, and immunohistochemistry. Vasospastic myocytes were depolarized and had a smaller contribution of GK toward maintenance of their membrane potential. Pharmacological block of Kv current in control myocytes mimicked the depolarization observed in vasospastic arteries. The degree of membrane depolarization was found to be compatible with the amount of vasoconstriction observed after SAH. We conclude that Kv2 dysfunction after SAH contributes to the pathogenesis of delayed cerebral vasospasm. This may confer a novel target for treatment of delayed cerebral vasospasm.

Expression and function of inwardly rectifying potassium channels after experimental subarachnoid hemorrhage

Cerebral vasospasm after subarachnoid hemorrhage (SAH) is because of smooth muscle contraction, although the mechanism of this contraction remains unresolved. Membrane potential controls the contractile state of arterial myocytes by gating voltage-sensitive calcium channels and is in turn primarily controlled by K+ ion conductance through several classes of K+ channels. We characterized the role of inwardly rectifying K+ (KIR) channels in vasospasm. Vasospasm was created in dogs using the double-hemorrhage model of SAH. Electrophysiological, real-time quantitative reverse-transcriptase polymerase chain reaction, Western blotting, immunohistochemistry, and isometric tension techniques were used to characterize the expression and function of KIR channels in normal and vasospastic basilar artery 7 days after SAH. Subarachnoid hemorrhage resulted in severe vasospasm of the basilar artery (mean of 61% ± 5% reduction in diameter). Membrane potential of pressurized vasospastic basilar arteries was significantly depolarized compared with control arteries (+-46 ± 1.4 mV versus −29.8 ± 1.8 mV, respectively, P < 0.01). In whole-cell patch clamp of enzymatically isolated basilar artery myocytes, average KIR conductance was 1.6 ± 0.5 pS/pF in control cells and 9.2 ± 2.2 pS/pF in SAH cells (P = 0.007). Blocking KiR channels with BaCI2 (0.1 mmol/L) resulted in significantly greater membrane depolarization in vasospastic compared with normal myocytes. Expression of KIR 2.1 messenger ribonucleic acid (mRNA) was increased after SAH. Western blotting and immunohistochemistry also showed increased expression of KIR protein in vasospastic smooth muscle. Blockage of KIR channels in arteries under isometric tension produced a greater contraction in SAH than in control arteries. These results document increased expression of KIR 2.1 mRNA and protein during vasospasm after experimental SAH and suggest that this increase is a functionally significant adaptive response acting to reduce vasospasm.

Angiographic, hemodynamic and histological characterization of an arteriovenous fistula in rats

SummaryBackground. Our understanding of the pathogenesis of arteriovenous malformations (AVMs) and arteriovenous fistulas (AVFs) has been limited by the lack of adequate animal models. In this study we evaluate the time course of angiographic, hemodynamic and histopathological changes in an arteriovenous fistula in rats as a potential model. Methods. An arteriovenous fistula was created by a side-to-end anastomosis of the common carotid artery (CCA) to the external jugular vein (EJV). The animals underwent angiography of the fistula and were sacrificed 1, 7, 21, 42 or 90 days later. Flow and pressure measurements were performed in the CCA and ipsi- and contralateral EJV and detailed histological examination of whole mount sections of the fistula and cranium were done on fixed sections. Immunohistochemistry for CD31, smooth muscle α-actin and Ki-67 were performed. Findings. Hemodynamic changes occur immediately after fistula formation creating a stable high flow, low resistant state. This induces a gradual increase in the inner diameter of the EJV and transverse sinus followed by a decrease in size of the transverse sinus. This decrease is associated with increased expression of α-actin in the wall of the sinus. The fistula becomes angiographically and histologically stable after 21 days. Conclusion. This model describes the time course of hemodynamic and histopathological changes after occur after AVF formation. Stabilization after 21 days makes it an attractive model for mechanistic and therapeutic studies of AVFs.

Effects of a nitric oxide donor on and correlation of changes in cyclic nucleotide levels with experimental vasospasm

OBJECTIVEVasospasm after subarachnoid hemorrhage (SAH) may result from hemoglobin-mediated removal of nitric oxide (NO) from the arterial wall. We tested the ability of the long-acting, water-soluble, NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-1,2-diolate (DETA/NO), delivered via continuous intracisternal infusion, to prevent vasospasm in a nonhuman primate model of SAH. METHODSFirst, vasorelaxation in response to DETA/NO was characterized in vitro by using monkey basilar artery rings under isometric tension. Next, monkeys were randomized to undergo angiography, unilateral SAH, and no treatment (SAH only, n = 4) or treatment with DETA/NO (1 mmol/L, 12 ml/d, n = 4) or decomposed DETA/NO (at the same dose, n = 4). Vasospasm was assessed by angiography, which was performed on Day 0 and Day 7. Levels of cyclic adenosine monophosphate and cyclic guanosine monophosphate (cGMP) were measured in cerebral arteries on Day 7. RESULTSDETA/NO produced significant relaxation of monkey arteries in vitro, which reached a maximum at concentrations of 10−5 mol/L. In monkeys, angiography demonstrated significant vasospasm of the right intradural cerebral arteries in all three groups, with no significant difference in vasospasm among the groups (P > 0.05, analysis of variance). The ratios of cGMP or cyclic adenosine monophosphate levels in the right and left middle cerebral arteries were not different among the groups (P > 0.05, analysis of variance). There was no significant correlation between arterial cGMP contents and the severity of vasospasm. CONCLUSIONDETA/NO did not prevent vasospasm. There was no correlation between the severity of vasospasm and cyclic adenosine monophosphate and cGMP levels in the cerebral arteries. These results suggest that events downstream of cyclic nucleotides may be abnormal during vasospasm.

Intracisternal Sodium Nitroprusside Fails to Prevent Vasospasm in Nonhuman Primates

OBJECTIVE Hemoglobin contributes to vasospasm after subarachnoid hemorrhage. One mechanism may involve binding of nitric oxide, destruction of nitric oxide, or both. Support for this mechanism would be evidence that nitric oxide donors prevent vasospasm. This study attempted to provide such evidence. METHODS A randomized, blinded study was conducted in which 13 monkeys underwent cerebral angiography and creation of a right subarachnoid hemorrhage. Subcutaneous osmotic pumps were implanted to deliver sodium nitroprusside (n = 7) or vehicle (n = 6) via catheters into the right basal cisterns. Seven days later, angiography was repeated, and the animals were humanely killed. Levels of cyclic nucleotides, hemoglobins, and thiocyanate were measured. RESULTS Significant vasospasm of the right middle cerebral artery was present in animals treated with sodium nitroprusside (35 ± 22% reduction in diameter, P < 0.05, paired t test) and placebo (28 ± 20% reduction, P < 0.05, not significantly different from nitroprusside group by unpaired t test). Adequate delivery of sodium nitroprusside was supported by the finding of a significant increase in cyclic guanosine monophosphate levels in the cerebral arteries of treated animals compared with placebo (P < 0.05, unpaired t test). Thiocyanate was not present in significantly increased amounts in animals treated with nitroprusside, although this group did display elevated concentrations of nitrosyl hemoglobin (measured by electron paramagnetic resonance spectroscopy) and cyanomethemoglobin (measured by spectrophotometry) in the cerebrospinal fluid on Day 7. CONCLUSION The lack of effect of sodium nitroprusside was not the result of inadequate drug delivery because cyclic guanosine monophosphate levels were significantly increased in vasospastic arteries. Vasospasm may not have been prevented because of a toxic effect of sodium nitroprusside metabolites, involvement of smooth muscle relaxation or contraction processes downstream of cyclic guanosine monophosphate, or both.

Anterior interhemispheric approach for 100 tumors in and around the anterior third ventricle.

OBJECTIVE We report our experience with anterior interhemispheric approach for tumors in and around the anterior third ventricle, including surgical technique, instrumentation, pre- and postoperative hormonal disturbances, and resection rate. METHODS One hundred patients with 46 craniopharyngiomas, 12 hypothalamic gliomas, 12 meningiomas, 6 hypothalamic hamartomas, and 24 other lesions were operated on using an anterior interhemispheric approach with or without opening of the lamina terminalis. This surgical approach involves no frontal sinus opening; a narrow (approximately 15–20 mm in width) access between the bridging veins, which is sufficient to remove the tumor totally; and sparing of the anterior communicating artery. Specially designed long bipolar forceps and scissors are necessary for this approach, and concomitant use of angled instruments (endoscope, aspirator, and microforceps) is required frequently. The postsurgical follow-up period varied from 4 months to 18 years. RESULTS Total removal of the neoplasm was accomplished in 37 of 46 patients with craniopharyngiomas (80.4%), whereas subtotal resection was performed in hypothalamic gliomas. No significant differences in pre- and postoperative hormonal disturbances were observed in 37 craniopharyngiomas and 10 hypothalamic gliomas. There was no operative mortality. Visual acuity was preserved or improved in 68 of 75 patients assessed. The Karnofsky Performance Scale score did not deteriorate in 72 of 75 patients tested. CONCLUSION The minimally invasive anterior interhemispheric approach, with or without opening of the lamina terminalis, is useful for removal of tumors in and around the anterior third ventricle, such as craniopharyngiomas and hypothalamic gliomas.