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2-Arachidonoylglycerol (2-AG) is a major modulator of blood flow and platelet aggregation and a potential neuroprotectant. The present study investigated, for the first time, the effects of 2-AG on cerebral blood flow (CBF) in the first critical hours during middle cerebral artery occlusion (MCAO) and on platelet aggregation in rats. Adult male Sprague-Dawley rats ( n = 30) underwent permanent MCAO under isoflurane anesthesia and were randomly assigned to receive either 2-AG (6 mg/kg iv), monoacylglycerol lipase inhibitor JZL-184 (10 mg/kg iv), or vehicle ( n = 6 rats/group) treatment. CBF and cardiovascular responses were measured, by a blinded investigator, for up to 4 h. In separate experiments, platelet aggregation by 2-AG (19-300 µM) was assessed by whole blood aggregometry ( n = 40). 2-AG and JZL-184 significantly increased the severity of the CBF deficit versus vehicle (20.2 ± 8.8% and 22.7 ± 6.4% vs. 56.4 ± 12.1% of pre-MCAO baseline, respectively, P < 0.05) but had no effect on blood pressure or heart rate. While JZL-184 significantly increased the number of thrombi after MCAO, this did not reach significance by 2-AG. 2-AG induced platelet aggregation in rat whole blood in a similar manner to arachidonic acid and was significantly reduced by the cyclooxygenase inhibitors indomethacin and flurbiprofen and the thromboxane receptor antagonist ICI 192,605 ( P < 0.05). This is the first study showing that 2-AG increases the severity of the CBF deficit during MCAO, and further interrogation confirmed 2-AG-induced platelet aggregation in rats. These findings are important because 2-AG had previously been shown to exert neuroprotective actions and therefore force us to reevaluate the circumstances under which 2-AG is beneficial. NEW & NOTEWORTHY 2-Arachidonoylglycerol (2-AG) has neuroprotective properties; however, the present study revealed that 2-AG increases the severity of the cerebral blood flow deficit during middle cerebral artery occlusion in rats. Further interrogation showed that 2-AG induces platelet aggregation in rats. These findings force us to reevaluate the circumstances under which 2-AG is beneficial.
Assuntos
Ácidos Araquidônicos/toxicidade , Plaquetas/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Endocanabinoides/toxicidade , Glicerídeos/toxicidade , Infarto da Artéria Cerebral Média/sangue , Infarto da Artéria Cerebral Média/fisiopatologia , Fármacos Neuroprotetores/toxicidade , Agregação Plaquetária/efeitos dos fármacos , Animais , Plaquetas/metabolismo , Modelos Animais de Doenças , Masculino , Prostaglandina-Endoperóxido Sintases/sangue , Ratos Sprague-Dawley , Índice de Gravidade de Doença , Tromboxano A2/sangue , Fatores de TempoRESUMO
Dax-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital region on X-chromosome gene 1) blocks 17ß-estradiol biosynthesis and its knockdown would be expected to increase 17ß-estradiol production. We hypothesized that knockdown of Dax-1 in a conditionally immortalized neural stem cell (NSC) line, MHP36, is a useful approach to increase 17ß-estradiol production. Short hairpin (sh) RNA targeted to Dax-1 in NSCs, namely MHP36-Dax1KD cells, resulted in the degradation of Dax-1 RNA and attenuation of Dax-1 protein expression. In vitro, MHP36-Dax1KD cells exhibited overexpression of aromatase and increased 17ß-estradiol secretion compared to MHP36 cells. As 17ß-estradiol has been shown to promote the efficacy of cell therapy, we interrogated the application of 17ß-estradiol-enriched NSCs in a relevant in vivo disease model. We hypothesized that MHP36-Dax1KD cells will enhance functional recovery after transplantation in a stroke model. C57BL/6 male adult mice underwent ischemia/reperfusion by left middle cerebral artery occlusion for 45 min using an intraluminal thread. Two days later male mice randomly received vehicle, MHP36 cells, MHP36-Dax1KD cells, and MHP36 cells suspended in 17ß-estradiol (100 nm) or 17ß-estradiol alone (100 nm) with serial behavioral testing over 28 days followed by post-mortem histology and blinded analysis. Recovery of sensorimotor function was accelerated and enhanced, and lesion volume was reduced by MHP36-Dax1KD transplants. Regarding mechanisms, immunofluorescence indicated increased synaptic plasticity and neuronal differentiation after MHP36-Dax1KD transplants. In conclusion, knockdown of Dax-1 is a useful target to increase 17ß-estradiol biosynthesis in NSCs and improves functional recovery after stroke in vivo, possibly mediated through neuroprotection and improved synaptic plasticity. Therefore, targeting 17ß-estradiol biosynthesis in stem cells may be a promising therapeutic strategy for enhancing the efficacy of stem cell-based therapies for stroke.
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Stroke is an unmet clinical need with a paucity of treatments, at least in part because chronic stroke pathologies are prohibitive to 'first-generation' stem cell-based therapies. Hydrogels can remodel the hostile stroke microenvironment to aid endogenous and exogenous regenerative repair processes. However, no clinical trials have yet been successfully commissioned for these 'second-generation' hydrogel-based therapies for chronic ischaemic stroke regeneration. This review recommends a path forward to improve hydrogel technology for future clinical translation for stroke. Specifically, we suggest that a better understanding of human host stroke tissue-hydrogel interactions in addition to the effects of scaling up hydrogel volume to human-sized cavities would help guide translation of these second-generation regenerative stroke therapies.
Assuntos
Isquemia Encefálica , Acidente Vascular Cerebral , Humanos , Hidrogéis/farmacologia , Hidrogéis/uso terapêutico , Transplante de Células-Tronco , Acidente Vascular Cerebral/terapia , Engenharia TecidualRESUMO
BACKGROUND AND PURPOSE: To gain a better understanding of T cell behavior after stroke, we have developed real-time in vivo brain imaging of T cells by multiphoton microscopy after middle cerebral artery occlusion. METHODS: Adult male hCD2-GFP transgenic mice that exhibit green fluorescent protein-labeled T cells underwent permanent left distal middle cerebral artery occlusion by electrocoagulation (n=6) or sham surgery (n=6) and then multiphoton laser imaging 72 hours later. RESULTS: Extravasated T cell number significantly increased after middle cerebral artery occlusion versus sham. Two T cell populations existed after middle cerebral artery occlusion, possibly driven by 2 T cell subpopulations; 1 had significantly lower and the other significantly higher track velocity and displacement rate than sham. CONCLUSIONS: The different motilities and behaviors of T cells observed using our imaging approach after stroke could reveal important mechanisms of immune surveillance for future therapeutic exploitations.
Assuntos
Encéfalo/patologia , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Acidente Vascular Cerebral/patologia , Linfócitos T/patologia , Animais , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Infarto da Artéria Cerebral Média/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Acidente Vascular Cerebral/etiologiaRESUMO
Tissue-mimetic silk hydrogels are being explored for diverse healthcare applications, including stem cell delivery. However, the impact of stress relaxation of silk hydrogels on human mesenchymal stem cell (MSC) biology is poorly defined. The aim of this study was to fabricate silk hydrogels with tuned mechanical properties that allowed the regulation of MSC biology in two dimensions. The silk content and stiffness of both elastic and viscoelastic silk hydrogels were kept constant to permit direct comparisons. Gene expression of IL-1ß, IL-6, LIF, BMP-6, BMP-7, and protein tyrosine phosphatase receptor type C were substantially higher in MSCs cultured on elastic hydrogels than those on viscoelastic hydrogels, whereas this pattern was reversed for insulin, HNF-1A, and SOX-2. Protein expression was also mechanosensitive and the elastic cultures showed strong activation of IL-1ß signaling in response to hydrogel mechanics. An elastic substrate also induced higher consumption of glucose and aspartate, coupled with a higher secretion of lactate, than was observed in MSCs grown on viscoelastic substrate. However, both silk hydrogels changed the magnitude of consumption of glucose, pyruvate, glutamine, and aspartate, and also metabolite secretion, resulting in an overall lower metabolic activity than that found in control cells. Together, these findings describe how stress relaxation impacts the overall biology of MSCs cultured on silk hydrogels.
Assuntos
Fibroínas/química , Hidrogéis/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Animais , Bombyx/química , Técnicas de Cultura de Células/métodos , Proliferação de Células/efeitos dos fármacos , Módulo de Elasticidade , Expressão Gênica/efeitos dos fármacos , Humanos , RNA Mensageiro/metabolismo , Substâncias Viscoelásticas/químicaRESUMO
Silk has a long track record of use in humans, and recent advances in silk fibroin processing have opened up new material formats. However, these new formats and their applications have subsequently created a need to ascertain their biocompatibility. Therefore, the present aim was to quantify the haemocompatibility and inflammatory response of silk fibroin hydrogels. This work demonstrated that self-assembled silk fibroin hydrogels, as one of the most clinically relevant new formats, induced very low blood coagulation and platelet activation but elevated the inflammatory response of human whole blood in vitro. In vivo bioluminescence imaging of neutrophils and macrophages showed an acute, but mild, local inflammatory response which was lower than or similar to that induced by polyethylene glycol, a benchmark material. The time-dependent local immune response in vivo was corroborated by histology, immunofluorescence and murine whole blood analyses. Overall, this study confirms that silk fibroin hydrogels induce a similar immune response to that of PEG hydrogels, while also demonstrating the power of non-invasive bioluminescence imaging for monitoring tissue responses.
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Fibroínas , Animais , Materiais Biocompatíveis , Humanos , Hidrogéis , Imunidade Inata , Camundongos , SedaRESUMO
Assessment of outcome in preclinical studies of vascular cognitive impairment (VCI) is heterogenous. Through an ARUK Scottish Network supported questionnaire and workshop (mostly UK-based researchers), we aimed to determine underlying variability and what could be implemented to overcome identified challenges. Twelve UK VCI research centres were identified and invited to complete a questionnaire and attend a one-day workshop. Questionnaire responses demonstrated agreement that outcome assessments in VCI preclinical research vary by group and even those common across groups, may be performed differently. From the workshop, six themes were discussed: issues with preclinical models, reasons for choosing functional assessments, issues in interpretation of functional assessments, describing and reporting functional outcome assessments, sharing resources and expertise, and standardization of outcomes. Eight consensus points emerged demonstrating broadly that the chosen assessment should reflect the deficit being measured, and therefore that one assessment does not suit all models; guidance/standardisation on recording VCI outcome reporting is needed and that uniformity would be aided by a platform to share expertise, material, protocols and procedures thus reducing heterogeneity and so increasing potential for collaboration, comparison and replication. As a result of the workshop, UK wide consensus statements were agreed and future priorities for preclinical research identified.
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Demência Vascular , Modelos Animais de Doenças , Projetos de Pesquisa/normas , Animais , Consenso , Recuperação de Função Fisiológica , Inquéritos e Questionários , Reino UnidoRESUMO
Evidence exists for the potential protective effects of circulating ovarian hormones in stroke, and oestrogen reduces brain damage in animal ischaemia models. However, a recent clinical trial indicated that HRT (hormone-replacement therapy) increased the incidence of stroke in post-menopausal women, and detrimental effects of oestrogen on stroke outcome have been identified in a meta-analysis of HRT trials and in pre-clinical research studies. Therefore oestrogen is not an agent that can be promoted as a potential stroke therapy. Many published reviews have reported the neuroprotective effects of oestrogen in stroke, but have failed to include information on the detrimental effects. This issue is addressed in the present review, along with potential mechanisms of action, and the translational capacity of pre-clinical research.
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Estrogênios/uso terapêutico , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Acidente Vascular Cerebral/induzido quimicamente , Animais , Castração , Transtornos Cognitivos/induzido quimicamente , Doença das Coronárias/induzido quimicamente , Terapia de Reposição de Estrogênios/efeitos adversos , Estrogênios/efeitos adversos , Feminino , Humanos , Lipídeos/sangue , Masculino , Camundongos , Nootrópicos/uso terapêutico , Pós-Menopausa , Fatores de Risco , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/prevenção & controle , Trombose Venosa/induzido quimicamenteRESUMO
Targeting the brain cavity formed by an ischemic stroke is appealing for many regenerative treatment strategies but requires a robust delivery technology. We hypothesized that self-assembling silk fibroin hydrogels could serve as a reliable support matrix for regeneration in the stroke cavity. We therefore performed in vivo evaluation studies of self-assembling silk fibroin hydrogels after intracerebral injection in a rat stroke model. Adult male Sprague-Dawley rats (n = 24) underwent transient middle cerebral artery occlusion (MCAo) 2 weeks before random assignment to either no stereotaxic injection or a stereotaxic injection of either self-assembling silk fibroin hydrogels (4% w/v) or PBS into the lesion cavity. The impact on morbidity and mortality, space conformity, interaction with glial scar, interference with inflammatory response, and cell proliferation in the lesion cavity were examined for up to 7 weeks by a blinded investigator. Self-assembling hydrogels filled the stroke cavity with excellent space conformity and presented neither an overt microglial/macrophage response nor an adverse morbidity or mortality. The relationship between the number of proliferating cells and lesion volume was significantly changed by injection of self-assembling silk hydrogels. This in vivo stroke model confirmed that self-assembling silk fibroin hydrogels provide a favorable microenvironment as a future support matrix in the stroke cavity.
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Acute brain injury resulting from ischemic/hemorrhagic or traumatic damage is one of the leading causes of mortality and disability worldwide and is a significant burden to society. Neuroprotective options to counteract brain damage are very limited in stroke and traumatic brain injury (TBI). Given the multifaceted nature of acute brain injury and damage progression, several therapeutic targets may need to be addressed simultaneously to interfere with the evolution of the injury and improve the patient's outcome. Stem cells are ideal candidates since they act on various mechanisms of protection and repair, improving structural and functional outcomes after experimental stroke or TBI. Stem cells isolated from placenta offer advantages due to their early embryonic origin, ease of procurement, and ethical acceptance. We analyzed the evidence for the beneficial effects of placenta-derived stem cells in acute brain injury, with the focus on experimental studies of TBI and stroke, the engineering strategies pursued to foster cell potential, and characterization of the bioactive molecules secreted by placental cells, known as their secretome, as an alternative cell-free strategy. Results from the clinical application of placenta-derived stem cells for acute brain injury and ongoing clinical trials are summarily discussed.
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Lesões Encefálicas/terapia , Placenta/citologia , Medicina Regenerativa/métodos , Células-Tronco/citologia , Cordão Umbilical/citologia , Feminino , Humanos , Gravidez , Acidente Vascular Cerebral/terapiaRESUMO
Selective estrogen receptor (ER) agonists can indicate which receptor subtypes are implicated in neuroprotection. This study investigated the contribution of ERbeta, using the selective agonist diarylpropiolnitrile (DPN), in a rat model of stroke. Lister Hooded rats were ovariectomized and implanted with mini-pumps containing either DPN (8 mg kg(-1) day(-1)) (n=7) or vehicle (n=5). Sensorimotor function was assessed using a neurological score and the spontaneous forelimb use asymmetry (cylinder) test. One week later the animals received a middle cerebral artery occlusion (MCAO), and T(2)-weighted MRI at 48 h post-MCAO quantified ischemic damage. Functional recovery was tested for 7 days post-MCAO and brains processed for histological verification of infarct size. The MRI images revealed no significant differences in hemispheric lesion volumes between vehicle- and DPN-treated groups (35.6+/-3.5% and 30.8+/-1.7%, respectively [mean+/-SEM]; Student's unpaired t-test df=10, t=-1.357, p=0.453); this was confirmed histologically at 7 days. MCAO induced significant decline in neurological score performance (from 22 to 11 at 2 h post-MCAO) in the vehicle-treated animals, which was not significantly influenced by DPN. MCAO also induced significant changes in forelimb use in the cylinder test (10% reduction in contralateral, 20% reduction in both, and 30% increase in ipsilateral forelimb use) but this response was not significantly different between groups [F(1,1)=2.929, p=0.118, repeated-measures ANOVA]. In conclusion, pretreatment with the ERbeta agonist DPN did not influence infarct size or sensorimotor function in rats exposed to MCAO.
Assuntos
Antagonistas de Estrogênios/administração & dosagem , Infarto da Artéria Cerebral Média/prevenção & controle , Nitrilas/administração & dosagem , Propionatos/administração & dosagem , Análise de Variância , Animais , Modelos Animais de Doenças , Feminino , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Imageamento por Ressonância Magnética/métodos , Exame Neurológico , Ovariectomia/métodos , Desempenho Psicomotor/efeitos dos fármacos , Ratos , Índice de Gravidade de DoençaRESUMO
BACKGROUND: The effects of estrogen on endothelial function remain controversial. Endothelial function is perturbed in hypertension. We aimed to determine whether pre-existing hypertension can modify endothelial-dependent responses to estrogen. METHODS: We compared the effects of estrogen replacement on endothelial function in healthy female adult Wistar Kyoto (WKY) rats and stroke-prone spontaneously hypertensive rats (SHRSP). Basal and carbachol-stimulated nitric oxide (NO) bioavailability were studied in carotid artery rings in ovariectomized animals treated with estrogen or placebo for 2 weeks in vivo, or after 1 h of incubation in vitro. Basal NO bioavailability was defined as the increase in pressor responses in phenylephrine in the presence of NO synthase blockade. Superoxide (O(2)(-)) levels in aortas were measured by lucigenin chemiluminescence and endothelial NO synthase (eNOS) protein levels by Western blotting. RESULTS: Basal NO bioavailability was increased in WKY treated with estrogen for 2 weeks compared to placebo. In contrast, no change in NO bioavailability was observed in SHRSP. The O(2)(-) levels were higher in SHRSP than in WKY but unaffected by estrogen treatment in either strain. In WKY, but not in SHRSP, estrogen caused upregulation of eNOS. Similarly in vitro exposure to estrogen increased NO bioavailability in WKY but had no effect in SHRSP. In WKY, co-exposure to estrogen and LY294002, a PI3 kinase inhibitor, abrogated the effect of estrogen. CONCLUSIONS: The inability of estrogen to improve endothelial function in SHRSP may relate to a defect in eNOS activation pathways in this hypertensive rat strain.
Assuntos
Estrogênios/farmacologia , Óxido Nítrico/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Estrogênios/sangue , Feminino , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Óxido Nítrico Sintase/metabolismo , Fenilefrina/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptores de Estrogênio/metabolismo , Superóxidos/metabolismoRESUMO
Oestrogen is a complex hormone whose role as a neuroprotectant in experimental stroke has been published in numerous studies. However, although some clinical studies report a reduction in stroke incidence by oestrogen replacement therapy in postmenopausal women, others have found increased mortality and morbidity after stroke. Diathermy occlusion of the proximal middle cerebral artery (MCAO), one of the most reproducible rodent stroke models, has consequently been employed to investigate physiologic and supraphysiologic doses of 17beta-oestradiol on ischaemic brain damage. Lister Hooded rats were ovariectomised (OVX) and a 21-day release pellet (placebo, 0.025 or 0.25 mg 17beta-oestradiol) implanted in the neck. At 2 weeks after OVX, animals underwent MCAO and were perfusion fixed 24 hours later. Neuronal perikaryal damage was assessed from haematoxylin and eosin-stained sections and in adjacent sections, axonal pathology was assessed with amyloid precursor protein and Neurofilament 200 (NF-200) immunohistochemistry. 17beta-Oestradiol induced a dose-dependent increase in neuronal perikaryal damage, 0.025 and 0.25 mg 17beta-oestradiol increased damage by 20% (P<0.05) and 27.5% (P<0.01) compared with placebo. 17beta-Oestradiol did not influence axonal pathology compared with placebo. Our results suggest that 17beta-oestradiol treatment can exacerbate brain damage in experimental stroke. Thus, further investigation into the role of oestrogen and the mechanisms which mediate its effects in stroke is required.
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Estradiol/efeitos adversos , Infarto da Artéria Cerebral Média/tratamento farmacológico , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Carbazóis/administração & dosagem , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Estradiol/administração & dosagem , Estradiol/sangue , Feminino , Imuno-Histoquímica , Infarto da Artéria Cerebral Média/patologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurônios/ultraestrutura , Ovariectomia , RatosRESUMO
INTRODUCTION: Numerous studies demonstrate oestrogen's neuroprotective effect in stroke models, although the mechanisms are unclear. Since oestrogen is an antioxidant, we tested the hypothesis that oestrogen reduces stroke-induced damage by reducing free radical damage, particularly lipid peroxidation. METHODS: Sprague-Dawley rats were ovariectomised and a 17beta-oestradiol (0.25 mg, 21 day release) or placebo pellet implanted subcutaneously. Two weeks later, permanent middle cerebral artery occlusion (MCAO) was induced by intraluminal filament. At 2 and 24 h post-MCAO, neurological deficits were assessed. At the 24 h end point, plasma oestradiol was measured and brain sections stained with haematoxylin and eosin or lipid peroxidation marker, 4-hydroxynonenol (4-HNE) immunohistochemistry carried out to measure infarct volume and volume of tissue displaying oxidative damage, respectively. RESULTS: Plasma 17beta-oestradiol in oestradiol and placebo groups was 72.6+/-38.0 and 9.3+/-7.4 pg/ml (mean+/-SD), respectively. Infarct volume was significantly increased (118%) with oestradiol treatment (oestradiol=124+/-84.5, placebo=57+/-46.4 mm3, mean+/-SD, P<0.05). The relationship between 4-HNE and infarct volume was significantly influenced by 17beta-oestradiol. Neurological deficits were similar between groups (oestradiol median=13, placebo=14, max score=33). CONCLUSION: Two week pre-treatment with a high physiological dose of 17beta-oestradiol increased infarct volume after permanent MCAO. Although contrary to our original hypothesis, this result demonstrates that oestrogen does have the capacity to promote detrimental actions in the stroke-injured brain. Given the wide use of oestrogen (contraception, osteoporosis and menopause), more research to clarify the influence of oestrogen on brain injury is urgently required.
Assuntos
Isquemia Encefálica/metabolismo , Infarto Cerebral/induzido quimicamente , Estradiol/efeitos adversos , Peroxidação de Lipídeos/efeitos dos fármacos , Degeneração Neural/induzido quimicamente , Estresse Oxidativo/efeitos dos fármacos , Aldeídos/metabolismo , Animais , Antioxidantes/efeitos adversos , Isquemia Encefálica/fisiopatologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Infarto Cerebral/metabolismo , Infarto Cerebral/fisiopatologia , Modelos Animais de Doenças , Progressão da Doença , Implantes de Medicamento , Estradiol/sangue , Estradiol/farmacologia , Feminino , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/fisiopatologia , Peroxidação de Lipídeos/fisiologia , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Exame Neurológico , Ovariectomia , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
The recent completion of the Human Genome Project provides the potential to advance our knowledge of pathogenesis and identify the gene(s) associated with particular diseases. However, using human DNA to correlate individual genomic variations with particular disorders such as stroke will be extremely challenging because of the large number of variables within an individual, and across different populations. Mutant animal models of stroke such as the stroke-prone spontaneously hypertensive rat (SHRSP) provide the scientist with genetic homogeneity, not possible within a human population, to aid our search for causative genes. This chapter describes the methods our group have employed to study the genetic heritability of stroke sensitivity in the SHRSP. Sections are included on quantitative trait loci, mapping, and congenic strain construction for the identification of genetic determinants of stroke sensitivity in the SHRSP.
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Acidente Vascular Cerebral/genética , Animais , Cruzamentos Genéticos , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Infarto da Artéria Cerebral Média/etiologia , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/patologia , Masculino , Repetições de Microssatélites , Fenótipo , Reação em Cadeia da Polimerase , Locos de Características Quantitativas , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos Mutantes , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/patologiaRESUMO
Blood brain barrier (BBB) breakdown and neuroinflammation are key events in ischemic stroke morbidity and mortality. The present study investigated the effects of mast cell deficiency and stabilization on BBB breakdown and neutrophil infiltration in mice after transient middle cerebral artery occlusion (tMCAo). Adult male C57BL6/J wild type (WT) and mast cell-deficient (C57BL6/J Kit(Wsh/Wsh) (Wsh)) mice underwent tMCAo and BBB breakdown, brain edema and neutrophil infiltration were examined after 4 hours of reperfusion. Blood brain barrier breakdown, brain edema, and neutrophil infiltration were significantly reduced in Wsh versus WT mice (P<0.05). These results were reproduced pharmacologically using mast cell stabilizer, cromoglycate. Wild-type mice administered cromoglycate intraventricularly exhibited reduced BBB breakdown, brain edema, and neutrophil infiltration versus vehicle (P<0.05). There was no effect of cromoglycate versus vehicle in Wsh mice, validating specificity of cromoglycate on brain mast cells. Proteomic analysis in Wsh versus WT indicated that effects may be via expression of endoglin, endothelin-1, and matrix metalloproteinase-9. Using an in vivo model of mast cell deficiency, this is the first study showing that mast cells promote BBB breakdown in focal ischemia in mice, and opens up future opportunities for using mice to identify specific mechanisms of mast cell-related BBB injury.
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Barreira Hematoencefálica/fisiopatologia , Isquemia Encefálica/imunologia , Isquemia Encefálica/fisiopatologia , Mastócitos/patologia , Infiltração de Neutrófilos , Animais , Barreira Hematoencefálica/imunologia , Encéfalo/irrigação sanguínea , Encéfalo/imunologia , Encéfalo/fisiopatologia , Edema Encefálico/etiologia , Edema Encefálico/imunologia , Edema Encefálico/fisiopatologia , Isquemia Encefálica/complicações , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/imunologia , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
We previously reported that during pro-estrus (high endogenous estrogen levels), brain damage after middle cerebral artery occlusion (MCAO) was reduced in stroke-prone spontaneously hypertensive rats (SHRSP) but not in normotensive Wistar Kyoto rat (WKY). In the present study, we examined the effect of exogenous estrogen on brain damage after MCAO in SHRSP and WKY. A 17beta-estradiol (0.025 mg or 0.25 mg, 21 day release) or matching placebo pellet was implanted into ovariectomized WKY and SHRSP (3 to 4 months old) who then underwent distal diathermy-induced MCAO 2 weeks later. Plasma 17beta-estradiol levels for placebo and 17beta-estradiol groups were as follows: WKY 0.025 mg 16.4 +/- 8.5 (pg/mL, mean +/- SD) and 25.85 +/- 12.6; WKY 0.25 mg 18.2 +/- 9.0 and 69.8 +/- 27.4; SHRSP 0.25 mg 20.7 +/- 8.8 and 81.0 +/- 16.9. In SHRSP, infarct volumes at 24 hours after MCAO were similar in placebo and 17beta-estradiol groups: SHRSP 0.025 mg 126.7 +/- 15.3 mm (n = 6) and 114.0 +/- 14.1 mm (n = 8) (not significant); SHRSP 0.25 mg 113.5 +/- 22.3 mm (n = 8) and 129.7 +/- 26.2 mm (n = 7) (not significant), respectively. In WKY, 17beta-estradiol significantly increased infarct volume by 65% with 0.025 mg dose [36.1 +/- 20.7 mm (n = 8) and 59.7 +/- 19.3 mm (n = 8) (P = 0.033, unpaired t-test)] and by 96% with 0.25 mg dose [55.9 +/- 36.4 mm (n = 8) and 109.7 +/- 6.7 mm (n = 4) (P = 0.017)]. Thus, 17beta-estradiol increased stroke damage in normotensive rats with no significant effect in stroke-prone rats. Despite being contrary to our hypothesis, our findings add substance to the recently reported negative effects of 17beta-estradiol in clinical studies.
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Encéfalo/patologia , Estradiol/farmacologia , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/patologia , Animais , Peso Corporal , Encéfalo/efeitos dos fármacos , Modelos Animais de Doenças , Estradiol/sangue , Feminino , Humanos , Infarto da Artéria Cerebral Média , Ovariectomia , Placebos , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
As one of the leading causes of death within both the developed and developing world, stroke is a worldwide problem. Risk factors can be identified and controlled at the level of lifestyle changes; however, genetic components of stroke have yet to be identified. The identification of such genetic components is critical in the understanding, diagnosis, and treatment of stroke in the future. This review focuses on the genetic determinants of stroke in both human and experimental systems. Mendelian disorders, candidate genes, and twin studies provide evidence for a strong genetic component of stroke. Genome-wide scanning in both human and animal models has led to the identification of regions of the genome that contain genes for stroke susceptibility and sensitivity. Animal models of stroke allow for environmental control and genetic homogeneity, not possible within a human population, and therefore are essential for the dissection of this complex, multifactorial disorder. Future genetic and genomic strategies and their role in ultimate causative gene identification are discussed.
Assuntos
Acidente Vascular Cerebral/genética , Animais , Apolipoproteínas E/genética , Ligação Genética , Predisposição Genética para Doença , Humanos , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo III , Linhagem , Sistema Renina-Angiotensina/genética , Fatores de Risco , Acidente Vascular Cerebral/patologia , GêmeosRESUMO
This article reviews genetic studies of stroke, bearing insight into human evidence by using studies on twins as well as on monogenic forms of stroke. Focus will be given to inbred animal models that have been used to improve genetic homogeneity and to control environmental influences. Such animal studies have lead to the identification of quantitative trait loci harboring putative genes for stroke susceptibility and sensitivity. The major strategies for gene identification are discussed. Experimental animal models of stroke have also revealed a sexual dimorphism in stroke susceptibility and sensitivity and this article reviews 2 possible candidates, namely, the Y chromosome and estrogens.
Assuntos
Acidente Vascular Cerebral/genética , Gêmeos/genética , Animais , Modelos Animais de Doenças , Estrogênios/genética , Humanos , Fatores Sexuais , Estudos em Gêmeos como Assunto , Cromossomo Y/genéticaRESUMO
Rodent models of focal cerebral ischaemia are critical for understanding pathophysiological concepts in human stroke. The availability of genetically modified mice has prompted the adaptation of the intraluminal filament occlusion model of focal ischaemia for use in mice. In the present study, we investigated the effects of increasing duration of intraluminal occlusion on the extent and distribution of ischaemic pathology and local cerebral blood flow (LCBF) in C57Bl/6J mice, the most common background mouse strain. Volumetric assessment of ischaemic damage was performed after 15, 30 or 60 min occlusion followed by 24 h reperfusion. LCBF was measured after 15 and 60 min occlusion using quantitative 14C-iodoantipyrine autoradiography. The extent and distribution of ischaemic damage was highly sensitive to increasing occlusion duration. Recruitment of tissue outside MCA territory produced a steep increase in the volume of damage with increasing occlusion duration: 15 min (9+/-2 mm3); 30 min (56+/-6 mm3); 60 min (69+/-2 mm3). Significant increases in the severity of cerebral hypoperfusion were observed after 60 min compared to 15 min occlusion within and outside MCA territory, e.g. caudate nucleus (9+/-6 ml per 100 g per min at 60 min vs. 33 ml per 100 g per min at 15 min) and hippocampus (16+/-14 ml per 100 g per min at 60 min vs. 61+/-16 ml per 100 g per min at 15 min). MABP remained stable for 25 min after occlusion onset and declined thereafter. The integrity of the circle of Willis was examined by carbon black perfusion of the vasculature. A complete circle of Willis was present in only one of 10 mice. These results demonstrate that intraluminal filament occlusion in C57Bl/6J mice leads to an occlusion duration-dependent increase in severity of cerebral hypoperfusion and extension of ischaemic pathology beyond MCA territory.