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Gen Kusaka, Hitoshi Kimura, Ikuyo Kusaka, Eddie Perkins, Anil Nanda, and John H. Zhang

Object. Mitogen-activated protein kinase (MAPK) has been implicated in cerebral vasospasm after subarachnoid hemorrhage (SAH). This study was conducted to investigate whether Src tyrosine kinase, an upstream regulator of MAPK, is involved in cerebral vasospasm.

Methods. An established canine double-hemorrhage model was used. Twenty-four dogs were divided into four groups: control, vehicle-treated, Src inhibitor PP2—treated, and Src inhibitor damnacanthal—treated groups. Vehicle (dimethyl sulfoxide), PP2, or damnacanthal was injected daily into the cisterna magna of 18 dogs at 3 to 6 days after induction of SAH. Angiography was performed on Day 0 (the day on which the first blood injection was administered to induce SAH) and on Day 7. Western blot analysis of Src and MAPK activation in basilar arteries (BAs) collected on Day 7 post-SAH was performed.

Severe vasospasm was observed in the BAs of vehicle-treated dogs. Mild vasospasm was observed in all dogs treated with Src inhibitors. Phosphorylated Src and MAPK were increased after SAH and activation of these kinases in the BAs was abolished by PP2 and damnacanthal.

Conclusions. The tyrosine kinase Src is an important upstream regulator of MAPK, and inhibition of Src might offer a new therapy in the management of cerebral vasospasm.

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Eddie Perkins, Hitoshi Kimura, Andrew D. Parent, and John H. Zhang

Object. Whether cerebral vasospasm occurs only in surface vessels or also in parenchymal arterioles is debatable. The present study was undertaken to evaluate comprehensively the microvasculature of the brainstem after experimental subarachnoid hemorrhage (SAH).

Methods. Nine mongrel dogs of either sex, each weighing between 18 and 24 kg, underwent double blood injections spaced 48 hours apart; the injections were infused into the cisterna magna immediately after angiography of the basilar arteries (BAs). Three additional dogs assigned to a control group received no blood injections. The dogs were killed on Day 7. Axial sections obtained from the midpontine region of both control dogs and animals subjected to SAH were evaluated with respect to the morphological characteristics of vessels and neurons, and for ultrastructural changes.

Severe vasospasm occurred in the BAs of all dogs subjected to SAH. Nevertheless, in these animals, the luminal areas and vessel perimeter in parenchymal arterioles, but not in parenchymal venules, were observed to have increased when compared with those of control dogs (p < 0.01, t-test). No corrugation of the internal elastic lamina was observed and smooth-muscle and endothelial cells remained normal at the ultrastructural level in the dogs with SAH.

Conclusions. In this model, vasospasm of the BAs did not extend into the region of the pons to affect the intraparenchymal arterioles. Dilation of the parenchymal arterioles might serve as compensation for reduced blood flow. Thus, no neuronal ischemia or infarction resulted in the pontine region of the brain.

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Motoyoshi Satoh, Eddie Perkins, Hitoshi Kimura, Jiping Tang, Yi Chun, Donald D. Heistad, and John H. Zhang

Object. Gene transfer to cerebral vessels is a promising new therapeutic approach for cerebral vasospasm after subarachnoid hemorrhage (SAH). This study was undertaken to explore whether a delayed treatment with adenovirus encoding the prepro-calcitonin gene—related peptide (CGRP), 2 days after initial blood injection, reduces cerebral vasospasm in a double-hemorrhage model of severe vasospasm in dogs.

Methods. In 20 dogs, arterial blood was injected into the cisterna magna on Days 0 and 2. Thirty minutes after the second blood injection, the animals received either adenovirus encoding the prepro-CGRP gene (AdCMVCGRP—treated group, eight dogs) or adenovirus encoding the β-galactosidase gene (AdCMVβgal—treated group, six dogs) under the cytomegalovirus (CMV) promoter. One group of dogs did not receive treatment and served as controls (control SAH group, six dogs). Angiography was performed on Days 0 and 7 to assess cerebral vasospasm. On Day 7 following angiography, the animals were killed and their brains were stained with X-gal to detect the distribution of gene expression. Cerebrospinal fluid (CSF) was also tested for CGRP immunoreactivity.

Severe vasospasm was observed in control SAH dogs on Day 7, and the mean basilar artery (BA) diameter was 53.4 ± 5.5% of the value measured on Day 0. Treatment with AdCMVβgal did not alter vasospasm (the BA diameter was 55 ± 3.9% of that measured on Day 0). The leptomeninges and adventitia of the BAs of dogs treated using AdCMVβgal demonstrated positive staining with X-gal. High levels of CGRP were measured in CSF from dogs that received AdCMVCGRP. In the group treated with AdCMVCGRP, vasospasm was significantly reduced (the BA diameter was 78.2 ± 5.3% of that measured on Day 0, p < 0.05 compared with the control SAH group and the AdCMVβgal group).

Conclusions. In a model of severe vasospasm in dogs, gene transfer of CGRP after injection of blood attenuated cerebral vasospasm after SAH.

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Hitoshi Kimura, Toshinari Meguro, Ahmed Badr, and John H. Zhang

Object. The naphthylsulfonate derivative suramin is an inhibitor of growth factor receptors (receptor tyrosine kinases) and G protein—coupled P2Y receptors. Both types of these receptors are suspected of being involved in cerebral vasospasm after subarachnoid hemorrhage (SAH). In the current study, the authors examined the therapeutic effects of suramin and a selective P2X-receptor antagonist, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), in the reversal of vasospasm in an established canine double-hemorrhage model.

Methods. Twenty-four dogs underwent double blood injection into the cisterna magna, with injections given on Days 0 and 2. The dogs were divided randomly into three groups (six animals in each group) to be treated from Days 2 through 6 with the vehicle dimethyl sulfoxide, suramin, or PPADS. An additional group of six dogs received double blood injection without any treatment and served as an SAH control group. The animals were killed on Day 7. Angiography was performed on Day 0 before blood injection and again on Day 7 before the animals were killed. After the death of the animals, the basilar arteries (BAs) were collected for morphological studies and determination of tyrosine kinase expression, and the bloody cerebrospinal fluid (CSF) produced by the hemorrhages was collected for measurement of oxyhemoglobin and adenosine triphosphate (ATP).

In the SAH control group, the mean diameter of the BAs on Day 7 was 46.23 ± 6.32% of the value on Day 0 (which served as a reference of 100%). In the DMSO-treated group, the mean residual diameter of the BA was 47.77 ± 0.8% on Day 7 compared with the value on Day 0. Suramin, but not PPADS, increased the residual diameter to 74.02 ± 4.24% on Day 7. On Day 7 the level of ATP in the CSF was decreased and the level of oxyhemoglobin was increased, compared with values measured on Day 0. Suramin, but not PPADS, reduced tyrosine phosphorylation in the spastic BAs.

Conclusions. By reducing tyrosine kinase activity, suramin may be useful in the treatment of cerebral vasospasm.