RESUMO
OBJECTIVE: Worldwide, cerebral vasospasm after subarachnoid hemorrhage (SAH) has an estimated morbidity and mortality of 1.2 million annually. While it has long been suspected that reactive oxygen species play a major role in the etiology of cerebral vasospasm after SAH, promising results in animal work were not borne out in human clinical trials, despite intensive research effort. The purpose of this study is to investigate the role of glutathione peroxidase in the SAH cerebrospinal fluid milieu. METHODS: We utilized commercially available kits for the quantitation of glutathione peroxidase 1 (glutathione peroxidase) activity and oxygen radical capacity and sodium dodecyl sulfate polyacrylamide gel electrophoresis with Western blotting with specific antibodies to human glutathione peroxidase to determine the enzyme content of the cerebrospinal fluid samples. Human cerebrospinal fluid was obtained in an Institutional Review Board-exempt manner for this study in the following groups: control (no SAH), CSF(C) (SAH but no vasospasm on angiography) and CSF(V) (SAH with clinical and angiographic vasospasm). RESULTS: We found that glutathione peroxidase activity is significantly higher in CSF(V) compared with CSF(C), and this is reflected in a higher total oxidative capacity in CSF(V). Despite similar levels of glutathione peroxidase protein, CSF(V) had significantly greater activity than CSF(C). DISCUSSION: These results further elucidate previous research from this laboratory, showing increased oxidative stress in CSF(V) compared with CSF(C). In conclusion, there appears to be increased glutathione peroxidase activity in CSF(V), despite there being increased levels of oxidative stress markers, suggesting overwhelming oxidative stress may play a role in cerebral vasospasm after SAH.
Assuntos
Glutationa Peroxidase/líquido cefalorraquidiano , Estresse Oxidativo , Hemorragia Subaracnóidea/líquido cefalorraquidiano , Hemorragia Subaracnóidea/complicações , Vasoespasmo Intracraniano/líquido cefalorraquidiano , Vasoespasmo Intracraniano/etiologia , Adulto , Idoso , Análise de Variância , Angiografia/métodos , Antioxidantes/metabolismo , Colorimetria/métodos , Feminino , Humanos , Peroxidação de Lipídeos/fisiologia , Masculino , Malondialdeído/líquido cefalorraquidiano , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio , Glutationa Peroxidase GPX1RESUMO
Cerebral vasospasm (CV) remains a significant cause of delayed neurological deficit and ischemic damage after subarachnoid hemorrhage (SAH), despite intensive research effort. The current lack of an effective therapeutic approach is somewhat due to our lack of understanding regarding the mechanism by which this pathological constriction develops. Recent evidence implicates bilirubin oxidation products (BOXes) in the etiology of CV after SAH: BOXes are found in cerebrospinal fluid from SAH patients with symptomatic or angiographically visible vasospasm (CSFV) but not in CSF from SAH patients with no vasospasm (CSFC). We have previously published research suggesting that the etiology of CV comprises two components: a physiological stimulation to constrict and a pathological failure to relax. Both these components are elicited by CSFV, but not CSFC, and BOXes synthesized in the laboratory potentiate physiological constriction in arterial smooth muscle in vitro, and elicit contraction in pial arteries in vivo. In this paper, we will present our results concerning the action of BOXes on arterial smooth muscle constriction, compared with CSFV. We will also present evidence implicating temporal changes in PKC isoforms and Rho expression in both BOXes- and CSFV-elicited smooth muscle responses.
Assuntos
Bilirrubina/metabolismo , Músculo Liso Vascular/metabolismo , Proteína Quinase C/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Artérias Cerebrais/patologia , Hemorragia , Modelos Biológicos , Oxigênio/metabolismo , Hemorragia Subaracnóidea/patologia , Suínos , Fatores de Tempo , Vasoespasmo Intracraniano/patologiaRESUMO
The goal of this study was to determine whether targeted, Rhodamine-labeled echogenic liposomes (Rh-ELIP) containing nanobubbles could be delivered to the arterial wall, and whether 1-MHz continuous wave ultrasound would enhance this delivery profile. Aortae excised from apolipoprotein-E-deficient (n=8) and wild-type (n=8) mice were mounted in a pulsatile flow system through which Rh-ELIP were delivered in a stream of bovine serum albumin. Half the aortae from each group were treated with 1-MHz continuous wave ultrasound at 0.49 MPa peak-to-peak pressure, and half underwent sham exposure. Ultrasound parameters were chosen to promote stable cavitation and avoid inertial cavitation. A broadband hydrophone was used to monitor cavitation activity. After treatment, aortic sections were prepared for histology and analyzed by an individual blinded to treatment conditions. Delivery of Rh-ELIP to the vascular endothelium was observed, and sub-endothelial penetration of Rh-ELIP was present in five of five ultrasound-treated aortae and was absent in those not exposed to ultrasound. However, the degree of penetration in the ultrasound-exposed aortae was variable. There was no evidence of ultrasound-mediated tissue damage in any specimen. Ultrasound-enhanced delivery within the arterial wall was demonstrated in this novel model, which allows quantitative evaluation of therapeutic delivery.
Assuntos
Aorta/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Lipossomos/administração & dosagem , Ultrassom , Animais , Aorta/diagnóstico por imagem , Aorta/patologia , Apolipoproteínas E/genética , Apolipoproteínas E/fisiologia , Desenho de Equipamento , Feminino , Técnicas In Vitro , Bicamadas Lipídicas/metabolismo , Lipossomos/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Modelos Animais , Rodaminas/química , Espectrometria de Fluorescência , UltrassonografiaRESUMO
INTRODUCTION: Cerebral vasospasm after subarachnoid hemorrhage (SAH) is a serious complication resulting in delayed neurological deficit, increased morbidity, mortality, longer hospital stays, and rehabilitation time. It afflicts approximately 35 per 100,000 Americans per year, and there is currently no effective therapy. We present in vitro data suggesting that increasing intrinsic nitric oxide relaxation pathways in vascular smooth muscle via dopaminergic agonism ameliorates cerebral vasospasm after SAH. METHODS: Cerebrospinal fluid (CSF) from patients with cerebral vasospasm after SAH (CSF(V)) was used to induce vasospasm in porcine carotid artery in vitro. Dopamine was added to test its ability to reverse spasm, and specific dopamine receptor antagonists were used to determine which receptor mediated the protection. Immunohistochemical techniques confirmed the presence of dopamine receptor subtypes and the involvement of NOS in the mechanism of dopamine protection. RESULTS: Dopamine receptor 1, 2, and 3 subtypes are all present in porcine carotid artery. Dopamine significantly reversed spasm in vitro (67% relaxation), and this relaxation was prevented by Haloperidol, a D(2)R antagonist (10% relaxation, P < 0.05), but not by D(1) or D(3)-receptor antagonism. Both eNOS and iNOS expression were increased significantly in response to CSF(V) alone, and this was significantly enhanced by addition of dopamine, and blocked by Haloperidol. CONCLUSION: Cerebral vasospasm is significantly reversed in a functional measure of vasospasm in vitro by dopamine, via a D(2)R-mediated pathway. The increase in NOS protein seen in both the endothelium and vascular smooth muscle in response to CSF(V) is enhanced by dopamine, also in a D(2)R-dependent mechanism.