Kenji Kanamaru

Heme oxygenase-1 gene induction as an intrinsic regulation against delayed cerebral vasospasm in rats

Delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) causes cerebral ischemia and infarction. To date, the pathogenesis and gene expression associated with vasospasm remain poorly understood. The present study used fluorescent differential display to identify differentially expressed genes in a rat model of SAH. By using quantitative RT-PCR, we found that heme oxygenase-1 (HO-1) mRNA was prominently induced in the basilar artery and modestly in brain tissue in a rat vasospasm model. A significant correlation was observed between the degree of vasospasm and HO-1 mRNA levels in the basilar arteries exhibiting vasospasm. Intracisternal injection of antisense HO-1 oligodeoxynucleotide (ODN) significantly delayed the clearance of oxyhemoglobin and deoxyhemoglobin from the subarachnoid space and aggravated angiographic vasospasm. Antisense HO-1 ODN inhibited HO-1 induction in the basilar arteries but not in the whole brain tissue. This phenomenon was not observed in the nontreated, sense HO-1 ODN-treated, or scrambled ODN-treated arteries. We report the protective effects of HO-1 gene induction in cerebral vasospasm after SAH, a finding that should provide a novel therapeutic approach for cerebral vasospasm.

Anterior cervical vertebrectomy and interbody fusion for multi-level spondylosis and ossification of the posterior longitudinal ligament

Multi-level cervical spondylosis and ossification of the posterior longitudinal ligament (OPLL) are well-documented causes of myelopathy. The choice of surgical procedures remain controversial. Between January 1983 and December 1987, we have performed anterior cervical vertebrectomy in 45 patients with cervical myelopathy caused by multi-level spondylosis and OPLL. They consisted of 19 patients with cervical spondylosis, 12 with OPLL, and 14 with combined lesions of both cervical spondylosis and OPLL. There were 32 men and 13 women. The mean age was 55 years, ranging from 35 to 70 years. In all of our 45 patients, anterior vertebrectomy, discectomy, removal of posterior osteophytes and OPLL, and interbody fusion were done for progressive myelopathy refractory to conservative treatment. In 2 of 45 patients, 5 vertebral bodies were resected; in 3 patients, 4 vertebral bodies were resected; in 12 patients, 3 vertebral bodies were resected, in 19 patients, 2 vertebral bodies were resected; and in 9 patients, 1 vertebral body was resected. Thirty-nine of 45 patients (87%) had good results. Neurological signs did not improve in 5 patients (11%). One patient died because of agranulocytosis secondary to treatment with antibiotics. In conclusion, cervical cord compression caused by lesions located principally in the anterior aspect of the spinal canal may be completely relieved via anterior vertebrectomy, discectomy, removal of the calcified ligament, and fusion.

Mechanisms of Osteopontin-Induced Stabilization of Blood-Brain Barrier Disruption After Subarachnoid Hemorrhage in Rats

Background and Purpose— Osteopontin (OPN) is an inducible, multifunctional, extracellular matrix protein that may be protective against blood-brain barrier (BBB) disruption after subarachnoid hemorrhage (SAH). However, the protective mechanisms remain unclear. Methods— We produced the endovascular perforation model of SAH in rats and studied the time course of OPN induction in brains by Western blotting and immunofluorescence (n=50). Then, 34 rats were randomly assigned to sham (n=3), sham+OPN small interfering RNA (siRNA, n=3), SAH+negative control siRNA (n=14), and SAH+OPN siRNA (n=14) groups, and 109 rats were allocated to sham+vehicle (n=17), sham+recombinant OPN (n=17), SAH+vehicle (n=33), SAH+recombinant OPN (n=31), and SAH+recombinant OPN+l-arginyl-glycyl-l-aspartate motif-containing hexapeptide (n=11) groups. The effects of OPN siRNA or recombinant OPN on BBB disruption and related proteins were studied. Results— OPN was significantly induced in reactive astrocytes and capillary endothelial cells, peaking at 72 hours after SAH, during the recovery phase of BBB disruption. Blockage of endogenous OPN induction exacerbated BBB disruption and was associated with a reduction of angiopoietin-1 and mitogen-activated protein kinase (MAPK) phosphatase-1 (an endogenous MAPK inhibitor), activation of MAPKs, and induction of vascular endothelial growth factor-A at 72 hours after SAH, whereas recombinant OPN treatment improved it and was associated with MAPK phosphatase-1 induction, MAPK inactivation, and vascular endothelial growth factor-A reduction, which was blocked by l-arginyl-glycyl-l-aspartate motif-containing hexapeptide at 24 hours after SAH. Vascular endothelial growth factor-B and angiopoietin-2 levels were unchanged. Conclusions— OPN may increase MAPK phosphatase-1 that inactivates MAPKs, upstream and downstream of vascular endothelial growth factor-A, by binding to l-arginyl-glycyl-l-aspartate-dependent integrin receptors, suggesting a novel mechanism of OPN-induced post-SAH BBB protection.

Protective effects of recombinant osteopontin on early brain injury after subarachnoid hemorrhage in rats

Objective: Accumulated evidence suggests that the primary cause of poor outcome after subarachnoid hemorrhage is not only cerebral arterial narrowing but also early brain injury. Our objective was to determine the effect of recombinant osteopontin, a pleiotropic extracellular matrix glycoprotein, on early brain injury after subarachnoid hemorrhage in rats. Design: Controlled in vivo laboratory study. Setting: Animal research laboratory. Subjects: One hundred seventy-seven male adult Sprague-Dawley rats weighing 300 to 370 g. Interventions: The endovascular perforation model of subarachnoid hemorrhage was produced. Subarachnoid hemorrhage or sham-operated rats were treated with an equal volume (1 &mgr;L) of pre-subarachnoid hemorrhage intracerebroventricular administration of two dosages (0.02 and 0.1 &mgr;g) of recombinant osteopontin, albumin, or vehicle. Body weight, neurologic scores, brain edema, and blood–brain barrier disruption were evaluated, and Western blot analyses were performed to determine the effect of recombinant osteopontin on matrix metalloproteinase-9, substrates of matrix metalloproteinase-9 (zona occludens-1, laminin), tissue inhibitor of matrix metalloproteinase-1, inflammation (interleukin-1&bgr;), and nuclear factor-&kgr;B signaling pathways. Measurements and Main Results: Treatment with recombinant osteopontin prevented a significant loss in body weight, neurologic impairment, brain edema, and blood–brain barrier disruption after subarachnoid hemorrhage. These effects were associated with the deactivation of nuclear factor-&kgr;B activity, inhibition of matrix metalloproteinase-9 induction, the maintenance of tissue inhibitor of matrix metalloproteinase-1, the consequent preservation of the cerebral microvessel basal lamina protein laminin, and the tight junction protein zona occludens-1. Conclusions: These results demonstrate that recombinant osteopontin treatment is effective for early brain injury after subarachnoid hemorrhage.

Endothelium-dependent relaxation of canine basilar arteries. Part 2: Inhibition by hemoglobin and cerebrospinal fluid from patients with aneurysmal subarachnoid hemorrhage.

The effects of hemoglobin and cerebrospinal fluid from patients with subarachnoid hemorrhage (CSF-SAH) on endothelium-dependent relaxation were studied. At 10(-6) M, hemoglobin somewhat inhibited the endothelium-dependent relaxation induced by A23187 in rings of canine basilar artery. At 3 X 10(-6) M, it almost completely inhibited the same response. At 3 X 10(-6) M, hemoglobin did not significantly inhibit smooth muscle relaxation mechanisms as papaverine-induced relaxation was not inhibited by hemoglobin. It was also demonstrated that pretreatment of arterial rings with CSF-SAH resulted in a dose-dependent inhibition of relaxation induced by A23187. The inhibitory effect of CSF-SAH was prominent in the case in which a high oxyhemoglobin concentration was measured by spectrophotometry. Normal CSF from patients without SAH did not affect endothelium-dependent relaxation. These results suggest that hemoglobin released from lysed erythrocytes inhibits endothelium-dependent relaxation of canine basilar arteries and may also play an important role in the pathogenesis of cerebral vasospasm after aneurysmal subarachnoid hemorrhage.

Granulocyte-colony stimulating factor inhibits apoptotic neuron loss after neonatal hypoxia-ischemia in rats.

Neonatal hypoxia-ischemia (HI) is an important clinical problem with few effective treatments. Granulocyte-colony stimulating factor (G-CSF) is an endogenous peptide hormone of the hematopoietic system that has been shown to be neuroprotective in focal ischemia in vivo and is currently in phase I/II clinical trials for ischemic stroke in humans. We tested G-CSF in a rat model of neonatal hypoxia-ischemia in postnatal day 7 unsexed rat pups. Three groups of animals were used: hypoxia-ischemia (HI, n=67), hypoxia-ischemia with G-CSF treatment (HI+G, n=65), and healthy control (C, n=53). G-CSF (50 microg/kg, subcutaneous) was administered 1 h after HI and given on four subsequent days (five total injections). Animals were euthanized 24 h, 1, 2, and 3 weeks after HI. Assessment included brain weight, histology, immunohistochemistry, and Western blotting. G-CSF treatment was associated with improved quantitative brain weight and qualitative Nissl histology after hypoxia-ischemia. TUNEL demonstrated reduced apoptosis in group HI+G. Western blot demonstrated decreased expression of Bax and cleaved caspase-3 in group HI+G. G-CSF treatment was also associated with increased expression of STAT3, Bcl-2, and Pim-1, all of which may have participated in the anti-apoptotic effect of the drug. We conclude that G-CSF ameliorates hypoxic-ischemic brain injury and that this may occur in part by an inhibition of apoptotic cell death.

Neuromelanin magnetic resonance imaging in Parkinson's disease and multiple system atrophy.

Pigmented neurons in the substantia nigra pars compacta (SNc) and locus coeruleus (LC) show decreased numbers differentially in Parkinsons disease (PD) and multiple system atrophy (MSA). Recent reports have described that fast spin-echo T1-weighted magnetic resonance imaging (MRI) by a 3-tesla machine can visualize neuromelanin-related contrast of the noradrenergic and dopaminergic neurons respectively in the LC and the SNc. Using neuromelanin MRI at 3 T, we investigated possible alterations of these catecholaminergic neurons in 32 PD and 9 MSA patients, and compared the results with those of 23 normal volunteers. The contrast ratio of the LC and SNc was decreased in MSA and PD patients, most prominently in the LC in MSA patients. The contrast ratio of the SNc was correlated with the Hoehn-Yahr stage of PD and the severity of neuroradiological abnormalities in MSA. These results indicate a potential diagnostic value of neuromelanin MRI to distinguish MSA patients from normal and PD patients.

Recombinant Osteopontin in Cerebral Vasospasm After Subarachnoid Hemorrhage

Osteopontin (OPN), a pleiotropic extracellular matrix glycoprotein, has been reported to be protective against ischemic lesions, but effects of OPN on vascular functions have not been investigated. The aim of this study was to assess whether recombinant OPN (r‐OPN) could prevent cerebral vasospasm after subarachnoid hemorrhage (SAH) in rats.

Effect of Vasospasm on Heme Oxygenases in a Rat Model of Subarachnoid Hemorrhage

BACKGROUND AND PURPOSE Subarachnoid hemorrhage (SAH)-induced heme oxygenase-1 (HO-1) in glia throughout the rat brain without affecting heme oxygenase-2 (HO-2). However, the relationship between cerebral vasospasm and the expression of heme oxygenases after SAH is thus far unknown. The purpose of the present study was to clarify the effect of vasospasm on the expression of heme oxygenases in a rat model of SAH. METHODS Endothelin, hemolysate, hemolysate saturated with carbon monoxide (CO-hemolysate), and saline were injected into the cisterna magna of adult rats. Angiography was repeated before each injection and 15 and 60 minutes and 24 hours after each injection. Immunocytochemistry for HO-1, HO-2, and glial fibrillary acidic protein (GFAP) was performed 24 hours after the injection. RESULTS A significant vasospasm occurred in the basilar artery after the injection of endothelin, hemolysate, and CO-hemolysate. The degree of vasospasm was most prominent 15 minutes after each injection. There was no significant difference in the degree of vasospasm among injections. The HO-1 was induced exclusively in the glial cells throughout the brain after injection of hemolysate and CO-hemolysate; however, it was not induced by endothelin and saline. In the dentate gyrus of the hippocampus and the molecular layer of the cerebellum, the HO-1-positive cells were also stained for GFAP, suggesting astrocytic glial cells. On the other hand, HO-2 immunoreactivity was abundant in neurons and was not affected by endothelin, hemolysate, CO-hemolysate, or saline. CONCLUSIONS It is suggested that heme per se, rather than ischemia induced by vasospasm, plays a pivotal role in the expression of HO-1 in this rat model.

Effects of Nitroglycerin on Vasospasm and Cyclic Nucleotides in a Primate Model of Subarachnoid Hemorrhage

BACKGROUND AND PURPOSE Nitroglycerin, as well as nitric oxide, causes hyperpolarization and cGMP elevation in vascular smooth muscle cells. It is unknown whether nitroglycerin ameliorates vasospasm by an increase in cGMP levels after subarachnoid hemorrhage (SAH). The purpose of the present study was to measure the levels of both cGMP and cAMP in the cerebral arteries and parietal cerebral cortices in a primate model and to determine the effect of nitroglycerin on vasospasm after SAH. METHODS Chronic vasospasm was induced by clot placement around the right middle cerebral artery (MCA). Seven days after the surgery, angiography was repeated and either nitroglycerin (3 micrograms/kg per hour) or saline was administered intravenously. Angiography and regional cerebral blood flow (rCBF) measurements in the bilateral parietal cortices were performed before and after each treatment. Both cGMP and cAMP levels were measured in the cerebral arteries and bilateral parietal cortices. RESULTS A significant vasospasm occurred in the cerebral arteries on both sides, more prominently on the right side. Concomitantly, rCBF on the right side was significantly decreased (P < .05). In the right MCA, cGMP levels were significantly lower than in the normal MCA (P < .05). After the administration of nitroglycerin for 3 hours, the cerebral vessels were significantly dilated on both sides (P < .05), and rCBF was significantly increased on the right side (P < .05) but not on the left side. Although depressed cGMP levels in the right MCA were not recovered by nitroglycerin, a significant increase in cGMP levels was observed in the basilar artery (P < .05). In both parietal cortices, cGMP levels were significantly decreased after SAH (P < .05) and unchanged after nitroglycerin treatment. There were no significant changes in cAMP levels in SAH and after nitroglycerin treatment. CONCLUSIONS The vasodilator effect of nitroglycerin in spastic MCA may not be mediated by an increase in cGMP levels, suggesting an involvement of hyperpolarization of the smooth muscle cells. Given the increase in rCBF, nitroglycerin may be therapeutic for the treatment of vasospasm.