Norihito Shimamura

Inhibition of Integrin αvβ3 Ameliorates Focal Cerebral Ischemic Damage in the Rat Middle Cerebral Artery Occlusion Model

Background and Purpose— Recent studies have shown that selective inhibition of specific subsets of intercellular adhesion molecules protects the brain during ischemia. We studied selective inhibition of integrin αvβ3 with cyclo [Arg-Gly-Asp-d-Phe-Val] (cRGDfV) in the rat middle cerebral artery occlusion model (MCAO). Methods— Rats were treated before and after MCAO with cRGDfV. Physiological parameters, expression of integrin αvβ3, infarction volume, brain water content, Evans Blue exudation, IgG exudation, histology, immunohistochemistry, and western blotting were studied in 4 groups of animals: sham operation (n=13), untreated (n=18), nonfunctioning peptide treatment (n=19), and cRGDfV treatment (n=27). Results— Treatment with cRGDfV reduced infarction, reduced brain edema, reduced exudation of Evans blue and IgG, and prevented fibrinogen deposition. Western blotting showed reduction of phosphorylated Flk-1 (a vascular endothelial growth factor [VEGF] receptor), reduction of phosphorylated FAK (an intracellular kinase phosphorylated in the presence of VEGF), reduction of VEGF, and reduction of fibrinogen in the cRGDfV treatment group. Conclusions— The selective integrin αvβ3 inhibitor cRGDfV improves outcomes in the MCAO model by preserving the blood-brain barrier, which mechanistically may occur in a VEGF- and VEGF-receptor–dependent manner.

Comparison of silicon-coated nylon suture to plain nylon suture in the rat middle cerebral artery occlusion model

UNLABELLED A variety of intraluminal sutures have been used in the middle cerebral artery occlusion model (MCAO) of focal ischemia. In the present study we tested commercially available silicon-coated nylon suture in the MCAO model and compared the results to traditional monofilament nylon suture occlusion. Twelve Sprague-Dawley male rats were randomly divided two groups, MCAO with 4-0 nylon suture (Group N, n=6) and MCAO with silicone-coated 4-0 nylon suture (Group S, n=6). Rats were sacrificed 24 h after reperfusion. Assessment included mortality rates, neurological evaluation, and infarct volume. One rat died in each group from subarachanoid hemorrhage. Neurological evaluation demonstrated that Group S tended to have worse neurological outcomes than Group N, although this difference was not statistically significant. On TTC stain Group S had significantly larger infarct volumes than Group N. We conclude that the commercially available silicone-coated occlusion suture provides better occlusion of the middle cerebral artery than the traditional uncoated nylon suture. CLASSIFICATION Disease-related neuroscience (Section 6).

Effect of a free radical scavenger, edaravone, in the treatment of patients with aneurysmal subarachnoid hemorrhage.

OBJECTIVEIt is hypothesized that cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) is induced by free radicals released from a subarachnoid clot. This study therefore investigated the effect of a new free radical scavenger, edaravone, in the treatment of patients with aneurysmal SAH. METHODSNinety-one patients with aneurysmal SAH participated in this study and were randomized into a control group (n = 42) and an edaravone-treated group (n = 49). The difference between the 2 groups in terms of incidence of delayed ischemic neurological deficits (DINDs) and cerebral infarction caused by vasospasm, and Glasgow Outcome Scale score at 3 months after SAH were statistically analyzed. RESULTSThe incidence of DINDs was 21% in the control group and 10% in the edaravone-treated group, yet there was no statistically significant difference between the 2 groups (P = 0.118). In patients with DINDs, the incidence of cerebral infarction caused by vasospasm was 66% in the control group and 0% in the edaravone-treated group (P = 0.028), whereas the incidence of poor outcome caused by vasospasm was 71% in the control group and 0% in the edaravone-treated group (P = 0.046). CONCLUSIONWe found a trend toward a lesser incidence of DINDs and a lesser incidence of poor outcome caused by cerebral vasospasm in edaravone-treated patients. It might therefore be suggested that edaravone is a useful agent for the treatment of aneurysmal SAH.

Neurovascular and neuronal protection by E64d after focal cerebral ischemia in rats.

Calpains and cathepsins are two families of proteases that play an important role in ischemic cell death. In this study, we investigated the effect of E64d, a μ‐calpain and cathepsin B inhibitor, in the prevention of neuronal and endothelial apoptotic cell death after focal cerebral ischemia in rats. Rats underwent 2 hr of transient focal ischemia from middle cerebral artery occlusion (MCAO) and were sacrificed 24 hr later. E64d (5 mg/ kg intraperitoneally) was administered 30 min before MCAO. Assessment included neurological function, infarction volume, brain water content, blood–brain barrier permeability, histology, and immunohistochemistry. The E64d‐treated rats had significant brain protection against ischemic damage. We observed a reduction of infarction volume, brain edema, and improved neurological scores in E64d‐treated rats compared with the nontreated control. Furthermore, there was a remarkable reduction in both proteases and caspase‐3 activation and apoptotic changes in both neurons and endothelial cells in E64d‐treated rats. These results suggest that E64d protects the brain against ischemic/reperfusion injury by attenuating neuronal and endothelial apoptosis.

An in Vivo Quantifiable Model of Cochlear Neuronal Degeneration Induced by Central Process Injury

In the available in vivo experimental models for cochlear neuronal degeneration, the peripheral (hair cell side) process of the cochlear nerve has been injured in order to induce neuronal degeneration. However, there has been no dependable experimental model in which cochlear neuronal degeneration begins from the central (brain stem side) process. This lack of a central process injury model has probably been due to the experimental difficulties that had to be overcome in order to reproducibly and selectively injure the central process of the cochlear neurons while maintaining the patency of the internal auditory artery in small experimental animals such as rats. Using rats, we first developed a central process injury model in which the reduction of the spiral ganglion cells due to retrograde degeneration of cochlear neurons can be quantitatively evaluated. In our experimental model, the cochlear nerve was compressed and injured by a compression-recording (CR) electrode placed at the internal auditory meatus. First, the cochlear nerve was compressed until the compound action potentials of the cochlear nerve became flat, and then the CR electrode was advanced by various compression speeds (5, 10, or 200 micrometer/s) to reach the same depth (400 micrometer). In our model, therefore, the reduction of the spiral ganglion cells was caused compression speed dependently. This method made it possible to produce compression injury to the cochlear nerve without evidence of damage to the blood supply to the cochlea via the internal auditory artery. This model gives us the means to obtain knowledge that was previously impossible to derive from the peripheral process injury models.

Methylprednisolone ameliorates cochlear nerve degeneration following mechanical injury

We investigated whether methylprednisolone sodium succinate can ameliorate cochlear nerve degeneration following compression injury on the cerebellopontine angle portion of the cochlear nerve, using a quantitative animal experimental model that we have developed recently. In this model, cochlear nerve degeneration after compression could be quantitatively evaluated, while cochlear ischemia induced by the compression carefully maintained below the critical limit that causes irreversible damage to the cochlea. Eleven rats were treated with methylprednisolone during the pre- and post-compression period. Two weeks after compression, the numbers of SGC were compared between the rats that received the compression without and with methylprednisolone treatment. Methylprednisolone treatment improved the survival of SGC following cochlear nerve injury statistically highly significantly in the basal turn where the traumatic stress had been less than in the other cochlear turns in our experimental setting. Although it was not statistically significant, greater survival was also observed in the other cochlear turns. The results of this experimental study indicated that at least a portion of injured cochlear nerve had been potentially treatable, and that methylprednisolone might prevent such cochlear neurons from entering into the vicious process of irreversible damaging process.

Inhibition of integrin αvβ3 reduces blood-brain barrier breakdown in focal ischemia in rats

Ischemic stroke is a major cause of morbidity and mortality in industrialized nations. We tested the effect of postischemic treatment of cyclo‐RGDfV (cRGDfV), a selective inhibitor of integrin αvβ3, in the middle cerebral artery occlusion (MCAO) model of ischemic stroke in rats. Rats were randomly divided into three groups: sham operation (n = 13), MCAO with no treatment (n = 18), and MCAO with cRGDfV treatment (n = 28). Focal ischemia was induced with the suture occlusion method for 2 hr, and treatment was given 1 hr after reperfusion (3 hr after ischemia). All animals were sacrificed 24 hr after reperfusion. Assessment included neurological scores, infarction volumes, brain water content, Evans blue exudation, IgG exudation, histology, immunohistochemistry, and Western blotting. Treatment with cRGDfV ameliorated neurological deficits, reduced brain edema, and reduced exudation of Evans blue dye and IgG, but failed to reduce infarction volumes. Western blotting showed a reduction in phosphorylation of one subset of vascular endothelial growth factor (VEGF) receptors in the cRGDfV treatment group. Western blotting also demonstrated a significant reduction of fibrinogen in the cRGDfV treatment group. We conclude that poststroke treatment with cRGDfV reduces blood–brain barrier breakdown in focal ischemia, possibly through inhibition of VEGF‐mediated vascular breakdown.

Phenotypic Transformation of Smooth Muscle in Vasospasm after Aneurysmal Subarachnoid Hemorrhage

Differentiated smooth muscle cells (SMC) control vasoconstriction and vasodilation, but they can undergo transformation, proliferate, secret cytokines, and migrate into the subendotherial layer with adverse consequences. In this review, we discuss the phenotypic transformation of SMC in cerebral vasospasm after subarachnoid hemorrhage. Phenotypic transformation starts with an insult as caused by aneurysm rupture: Elevation of intracranial and blood pressure, secretion of norepinephrine, and mechanical force on an artery are factors that can cause aneurysm. The phenotypic transformation of SMC is accelerated by inflammation, thrombin, and growth factors. A wide variety of cytokines (e.g., interleukin (IL)-1β, IL-33, matrix metalloproteinases, nitric oxidase synthases, endothelins, thromboxane A2, mitogen-activated protein kinase, platelet-derived vascular growth factors, and vascular endothelial factor) all play roles in cerebral vasospasm (CVS). We summarize the correlations between various factors and the phenotypic transformation of SMC. A new target of this study is the transient receptor potential channel in CVS. Statin together with fasdil prevents phenotypic transformation of SMC in an animal model. Clazosentan prevents CVS and improves outcome in aneurysmal subarachnoid hemorrhage in a dose-dependent manner. Clinical trials of cilostazol for the prevention of phenotypic transformation of SMC have been reported, along with requisite experimental evidence. To conquer CVS in its complexity, we will ultimately need to elucidate its general, underlying mechanism.

Suppression of the Rho/Rho-Kinase Pathway and Prevention of Cerebral Vasospasm by Combination Treatment with Statin and Fasudil After Subarachnoid Hemorrhage in Rabbit

The Rho/Rho-kinase pathway is considered important in the pathogenesis of sustained smooth muscle cell contraction during cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH). The aims of this study were to investigate whether combination treatment, with pitavastatin as an inhibitor of RhoA and fasudil as an inhibitor of Rho-kinase, prevents the cerebral vasospasm. SAH was simulated using the double-hemorrhage rabbit model, and pitavastatin, or fasudil, or both (combination treatment) were administrated. The basilar artery (BA) cross-sectional area only in the combination treatment group was statistically larger than in the SAH group (p < 0.05). BA Rho-kinase, as measured by ELISA, was statistically reduced only in the combination treatment group compared with the SAH group (p < 0.05). In the other two treatment groups, pitavastatin or fasudil treatment group showed larger BA cross-sectional areas and lower value for BA Rho-kinase, but there were no statistically significant differences compared with the SAH group. The expression of endothelial nitric oxide synthase (eNOS), evaluated by immunohistochemistry in the pitavastatin group and the combination group, was higher than in the SAH group. Results indicate that combination treatment could extensively prevent cerebral vasospasm due to the synergic effect of combining pitavastatin and fasudil on the Rho/Rho-kinase pathway and on eNOS.

Apoptosis of auditory neurons following central process injury

Although apoptotic changes in auditory neurons induced by injury to peripheral processes (dendrites) have been intensively studied, apoptotic changes in auditory neurons induced by injury to central processes (axons of spiral ganglion cells, SGCs) have not been reported previously, probably due to lack of an experimental model. The present study reports for the first time the appearance, extent, and time course of SGC apoptosis following injury to the central processes. Apoptosis was studied in a rat model that consisted of compression of the auditory nerve in the cerebellopontine (CP) angle cistern with intraoperative recordings of auditory nerve compound action potentials (CAPs) to ensure highly reproducible results. Rats were killed between day 0 and day 14 after compression and apoptosis of SGCs was evaluated quantitatively as well as qualitatively by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining, anti-activated caspase-3 immunostaining, Hoechst 33342 staining, and electron microscopy. The average number of TUNEL-positive apoptotic SGCs in each cochlear turn increased from day 1 to day 5 and then decreased gradually to an undetectable level on day 14 after compression. The average proportion of apoptotic SGCs identified in any cochlear turn on any day was always lower than 10%. The results of our present study should be useful in determining the therapeutic time window for rescuing auditory neurons undergoing apoptosis due to injury during surgery in the CP angle.