Lateral Crônica Cranial Janela Preparação Ativa<em> In Vivo</em> Observação Após Distal Middle Cerebral Oclusão da Artéria em Ratos
Summary
oclusão cirúrgica de um ramo distal da artéria cerebral média (MCAo) é um modelo usado com freqüência em pesquisa acidente vascular cerebral experimental. Este manuscrito descreve a técnica básica de MCAo permanente, combinada com a inserção de uma janela craniana lateral, que oferece a oportunidade para microscopia intravital longitudinal em ratinhos.
Abstract
Isquemia cerebral focal (ou seja, acidente vascular cerebral isquémico) pode causar lesão cerebral principal, que conduz a uma perda grave da função neuronal e, consequentemente, a um hospedeiro de motor e deficiências cognitivas. Sua alta prevalência coloca um problema de saúde grave, como acidente vascular cerebral é uma das principais causas de incapacidade a longo prazo e de morte em todo o mundo 1. A recuperação da função neuronal é, na maioria dos casos, apenas parcial. Até agora, as opções de tratamento são muito limitadas, em particular devido à janela de tempo estreita para trombólise 2,3. Que determina os métodos para acelerar a recuperação de acidente vascular cerebral continua a ser um objectivo médico prime; No entanto, isto tem sido dificultado pela insuficiência de conhecimentos mecanísticos para o processo de recuperação. pesquisadores tempos experimentais frequentemente empregam modelos de roedores de isquémia cerebral focal. Para além da fase aguda, a investigação acidente vascular cerebral é cada vez mais centrada na fase sub-aguda e crónica após isquemia cerebral. A maioria dos pesquisadores AVC aplicar permanente ou tranSIENT oclusão da MCA em ratinhos ou ratos. Em pacientes, oclusões do MCA estão entre as causas mais frequentes de acidente vascular cerebral isquêmico 4. Além de oclusão proximal da MCA utilizando o modelo de filamento, oclusão cirúrgica do MCA distal é provavelmente o modelo mais utilizado na pesquisa acidente vascular cerebral experimental 5. Oclusão de um distal (à ramificação das artérias lenticulo-estriadas) MCA ramo normalmente poupa o estriado e afeta principalmente o neocórtex. oclusão do vaso pode ser permanente ou transitória. Alta reprodutibilidade do volume de lesão e as taxas de mortalidade muito baixas em relação ao resultado a longo prazo são as principais vantagens deste modelo. Aqui, demonstramos como realizar uma janela craniana (CW) preparação crônica lateral ao seio sagital e, posteriormente, como na cirurgia de induzir um acidente vascular cerebral distal debaixo da janela usando uma abordagem craniotomia. Esta abordagem pode ser aplicada para imagiologia sequencial de alterações agudas e crónicas após isquemia atravésepi-iluminação, confocal de varredura a laser, e de dois fótons de microscopia intravital.
Introduction
Stroke is among the principal causes of long-term disability and death worldwide1, coming second after coronary heart disease. In addition, stroke is the primary cause of long-term disability, underscoring its tremendous socioeconomic impact6-8. Beyond acute treatment, investigating new approaches and mechanisms to accelerate and enhance recovery after stroke remains a prime medical goal7.
In the last few decades, data from experimental stroke research has contributed substantially to understanding the complex pathophysiological cascades triggered by ischemia9,10. Excitotoxicity, apoptosis, peri-infarct depolarization, and inflammation have been identified as the most relevant mediators of cell death following focal cerebral ischemia. Moreover, using animal models of cerebral ischemia, important concepts, diagnostic modalities, and therapeutic approaches have been developed and validated (e.g., “penumbra” and thrombolysis)11.
The availability of experimental stroke models, combined with non-invasive imaging modalities (e.g., magnetic resonance imaging (MRI), computed tomography, or laser speckle contrast analysis), enables the researcher to investigate hyperacute and chronic pathophysiological changes induced by the ischemic insult in a longitudinal manner12. Along with studying the spatiotemporal profile of the evolving lesion, changes resembling neuronal plasticity can be investigated and correlated to functional outcomes and histological findings. Within the last few years, further methodological advances have been made using the combination of cerebral ischemia models and in vivo microscopy via cranial windows13. These new techniques allow investigators to analyze the neurovascular unit at the cellular and molecular level, with great analytic power in the acute, subacute, and chronic phases following focal cerebral ischemia14. Moreover, in vivo microscopy imaging of microcirculatory dynamics has revealed novel aspects of cerebral microvasculature function and angioarchitecture, with significant pathophysiological relevance15-17.
In this protocol, we present how to perform a chronic CW preparation lateral to the sagittal sinus and how to surgically induce a distal stroke underneath the window. This mouse model can be applied to sequential imaging of acute, subacute, and chronic changes following focal cerebral ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.
Protocol
Representative Results
Discussion
AVC é uma das principais causas de incapacidade a longo prazo e de morte em todo o mundo 1. Além do tratamento agudo, investigando novas abordagens e mecanismos para acelerar e melhorar a recuperação após acidente vascular cerebral continua a ser um objectivo primordial médica 7. pesquisadores tempos experimentais frequentemente empregam modelos de roedores de isquémia cerebral focal. De facto, os modelos indutores transiente ou permanente MCAo imitar um dos tipos mais comuns de isquemia cer…
Disclosures
The authors have nothing to disclose.
Acknowledgements
VP is a participant in the Charité Clinical Scientist Program, funded by the Charité – Universitätsmedizin Berlin and the Berlin Institute of Health. TB is an SNSF PostDoc Mobility fellow. The authors receive grant support from EinsteinStiftung/A-2012-153 to PV.
Materials
| Binocular surgical microscope | Zeiss | Stemi 2000 C | |
| Light source for microscope | Zeiss | CL 6000 LED | |
| Heating pad with rectal probe | FST | 21061-10 | |
| Stereotactic frame | Kopf | Model 930 | |
| Anaethesia system for isoflurane | Draeger | ||
| Isoflurane | Abott | ||
| Dumont forceps #5 | FST | 11251-10 | |
| Dumont forceps #7 | FST | 11271-30 | |
| Bipolar Forceps | Erbe | 20195-501 | |
| Bipolar Forceps | Erbe 20195-022 | ||
| Microdrill | FST 18000-17 | ||
| Needle holder | FST | 12010-14 | |
| 5-0 silk suture | Feuerstein, Suprama | ||
| 7-0 silk suture | Feuerstein,Suprama | ||
| 8-0 silk suture | Feuerstein, Suprama | ||
| Veterinary Recovery Chamber | Peco Services | V1200 | |
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