Lateral Crônica Cranial Janela Preparação Ativa<em> In Vivo</em> Observação Após Distal Middle Cerebral Oclusão da Artéria em Ratos
概要
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…
開示
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 | |
参考文献
- Mukherjee, D., Patil, C. G. Epidemiology and the global burden of stroke. World Neurosurg. 76 (6), 85-90 (2011).
- Ebinger, M., Prüss, H., et al. Effect of the use of ambulance-based thrombolysis on time to thrombolysis in acute ischemic stroke: a randomized clinical trial. JAMA. 311 (16), 1622-1631 (2014).
- Ebinger, M., Lindenlaub, S., et al. Prehospital thrombolysis: a manual from Berlin. J vis Exp. (81), e50534 (2013).
- Bogousslavsky, J., Van Melle, G., Regli, F. The Lausanne Stroke Registry: analysis of 1,000 consecutive patients with first stroke. Stroke. 19 (9), 1083-1092 (1988).
- Engel, O., Kolodziej, S., Dirnagl, U., Prinz, V. Modeling stroke in mice – middle cerebral artery occlusion with the filament model. J Vis Exp. (47), (2011).
- Donnan, G. A., Fisher, M., Macleod, M., Davis, S. M. Stroke. Lancet. 371 (9624), 1612-1623 (2008).
- Meairs, S., Wahlgren, N., et al. Stroke research priorities for the next decade–A representative view of the European scientific community. Cerebrovasc Dis. 22 (2-3), 75-82 (2006).
- Rosamond, W., Flegal, K., et al. Heart disease and stroke statistics–2007 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation. 115 (5), 69-171 (2007).
- Moskowitz, M. A., Lo, E. H., Iadecola, C. The science of stroke: mechanisms in search of treatments. Neuron. 67 (2), 181-198 (2010).
- Dirnagl, U., Iadecola, C., Moskowitz, M. A. Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci. 22 (9), 391-397 (1999).
- Dirnagl, U., Endres, M. Found in Translation: Preclinical Stroke Research Predicts Human Pathophysiology, Clinical Phenotypes, and Therapeutic Outcomes. Stroke. , (2014).
- Prinz, V., Hetzer, A. -. M., et al. MRI heralds secondary nigral lesion after brain ischemia in mice: a secondary time window for neuroprotection. J Cereb Blood Flow Metab. , (2015).
- Shih, A. Y., Mateo, C., Drew, P. J., Tsai, P. S., Kleinfeld, D. A polished and reinforced thinned-skull window for long-term imaging of the mouse brain. J Vis Exp. (61), (2012).
- Holtmaat, A., Bonhoeffer, T., et al. Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat Protoc. 4 (8), 1128-1144 (2009).
- Iadecola, C., Dirnagl, U. The microcircualtion–fantastic voyage: introduction. Stroke. 44 (6), 83 (2013).
- Blinder, P., Tsai, P. S., Kaufhold, J. P., Knutsen, P. M., Suhl, H., Kleinfeld, D. The cortical angiome: an interconnected vascular network with noncolumnar patterns of blood flow. Nat Neurosc. 16 (7), 889-897 (2013).
- Shih, A. Y., Driscoll, J. D., Drew, P. J., Nishimura, N., Schaffer, C. B., Kleinfeld, D. Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain. J Cereb Blood Flow Metab. 32 (7), 1277-1309 (2012).
- Cabrales, P., Carvalho, L. J. M. Intravital microscopy of the mouse brain microcirculation using a closed cranial window. J Vis Exp. (45), (2010).
- Rosell, A., Agin, V., et al. Distal occlusion of the middle cerebral artery in mice: are we ready to assess long-term functional outcome. Transl Stroke Res. 4 (3), 297-307 (2013).
- Dorand, R. D., Barkauskas, D. S., Evans, T. A., Petrosiute, A., Huang, A. Y. Comparison of intravital thinned skull and cranial window approaches to study CNS immunobiology in the mouse cortex. Intravital. 3 (2), (2014).
- Balkaya, M., et al. Assessing post-stroke behavior in mouse models of focal ischemia. J Cereb Blood Flow Metab. 33 (3), 330-338 (2013).
- Balkaya, M., Kröber, J., Gertz, K., Peruzzaro, S., Endres, M. Characterization of long-term functional outcome in a murine model of mild brain ischemia. J Neurosci Methods. 213 (2), 179-187 (2013).
- Freret, T., Bouet, V., et al. Behavioral deficits after distal focal cerebral ischemia in mice: Usefulness of adhesive removal test. Beh Neurosci. 123 (1), 224-230 (2009).
- Liu, S., Zhen, G., Meloni, B. P., Campbell, K., Winn, H. R. RODENT STROKE MODEL GUIDELINES FOR PRECLINICAL STROKE TRIALS (1ST EDITION). J Exp Stroke Trans Med. 2 (2), 2-27 (2009).
- Florian, B., Vintilescu, R., et al. Long-term hypothermia reduces infarct volume in aged rats after focal ischemia. Neurosci Lett. 438 (2), 180-185 (2008).
- Noor, R., Wang, C. X., Shuaib, A. Effects of hyperthermia on infarct volume in focal embolic model of cerebral ischemia in rats. Neurosci Lett. 349 (2), 130-132 (2003).
- Barber, P. A., Hoyte, L., Colbourne, F., Buchan, A. M. Temperature-regulated model of focal ischemia in the mouse: a study with histopathological and behavioral outcomes. Stroke. 35 (7), 1720-1725 (2004).
- Shin, H. K., Nishimura, M., et al. Mild induced hypertension improves blood flow and oxygen metabolism in transient focal cerebral ischemia. Stroke. 39 (5), 1548-1555 (2008).
- Kapinya, K. J., Prass, K., Dirnagl, U. Isoflurane induced prolonged protection against cerebral ischemia in mice: a redox sensitive mechanism. Neuroreport. 13 (11), 1431-1435 (2002).
- Gertz, K., Priller, J., et al. Physical activity improves long-term stroke outcome via endothelial nitric oxide synthase-dependent augmentation of neovascularization and cerebral blood flow. Circ Res. 99 (10), 1132-1140 (2006).
- Dirnagl, U. Bench to bedside: the quest for quality in experimental stroke research. J Cereb Blood Flow Metab. 26 (12), 1465-1478 (2006).
