Lateral Kronisk Kranie Window Forberedelse Aktiverer<em> In vivo</em> Observation Efter Distal mellem-cerebral arterieokklusion i mus
Summary
Kirurgisk okklusion af en distal midtcerebralarterie gren (MCAo) er et hyppigt anvendt model i eksperimentel slagtilfælde forskning. Dette håndskrift beskriver den grundlæggende teknik for permanent MCAO kombineret med indsættelse af en lateral kraniel vindue, der giver mulighed for langsgående intravital mikroskopi i mus.
Abstract
Fokal cerebral iskæmi (dvs. iskæmisk slagtilfælde) kan medføre stor hjerneskade, hvilket fører til et tab af neuronal funktion og følgelig med en masse motoriske og kognitive handicap. Dens høje forekomst udgør en alvorlig sundheds- byrde, som slagtilfælde er blandt de vigtigste årsager til langvarig invaliditet og død på verdensplan en. Inddrivelse af neuronal funktion er, i de fleste tilfælde, kun delvis. Indtil videre er behandlingsmuligheder meget begrænsede, især på grund af den snævre tidsvindue for trombolyse 2,3. Bestemmelse metoder til at fremskynde helbredelse fra slagtilfælde fortsat en førsteklasses medicinsk mål; Dette har dog været hæmmet af utilstrækkelige mekanistiske indsigt i helbredelsesprocessen. Eksperimentelle slagtilfælde forskere ofte anvender gnaver modeller af fokal cerebral iskæmi. Ud over den akutte fase, er slagtilfælde forskning i stigende grad fokuseret på sub-akut og kronisk fase efter cerebral iskæmi. De fleste takts forskere anvender permanent eller tranSIENT okklusion af MCA hos mus eller rotter. Hos patienter, tilstopninger af MCA er blandt de hyppigste årsager til iskæmisk slagtilfælde 4. Udover proximal okklusion af MCA hjælp glødetråden model, kirurgisk okklusion af den distale MCA er sandsynligvis den mest brugte model i eksperimentel slagtilfælde forskning 5. Okklusion af en distal (til forgreningen af lenticulo-striate arterier) MCA gren reservedele typisk striatum og primært påvirker neocortex. Vessel okklusion kan være permanent eller forbigående. Høj reproducerbarhed læsionsvolumen og meget lavere dødelighed i forhold til den langsigtede resultat er de vigtigste fordele ved denne model. Her viser vi, hvordan man udfører en kronisk kraniel vindue (CW) forberedelse lateralt til sinus sagittalis, og bagefter hvordan man kirurgisk inducere en distal slagtilfælde under vinduet ved anvendelse af en kraniotomi tilgang. Denne fremgangsmåde kan anvendes til sekventiel billeddannelse af akutte og kroniske forandringer efter iskæmi viaepi-belysning, konfokal laserscanning, og to-foton intravital mikroskopi.
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
Slagtilfælde er blandt de vigtigste årsager til langvarig invaliditet og død på verdensplan en. Beyond akut behandling, undersøger nye metoder og mekanismer til at fremskynde og forbedre inddrivelsen efter slagtilfælde er stadig en førsteklasses medicinsk mål 7. Eksperimentelle slagtilfælde forskere ofte anvender gnaver modeller af fokal cerebral iskæmi. Faktisk modeller inducerer forbigående eller permanent MCAo efterligner en af de mest almindelige typer af fokal cerebral iskæmi hos p…
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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