Summary

Lateral Kronisk Kranie Window Forberedelse Aktiverer<em> In vivo</em> Observation Efter Distal mellem-cerebral arterieokklusion i mus

Published: December 29, 2016
doi:

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

ETIK ERKLÆRING: Eksperimenter med forsøgsdyr blev udført i overensstemmelse med de retningslinjer og bestemmelser fastsat af Landesamt fuer Gesundheit und Soziales, Berlin, Tyskland (G0298 / 13) og de ANKOMMER kriterier, som er relevant. Til denne undersøgelse 10- til 12-uger gamle C57BL / 6J mus blev anvendt. 1. Lateral Kronisk Kranie Window Forberedelse Udfør anæstesi med en subkutan injektion af ketamin (90 mg / kg) og xylazin (10 mg / kg). Test for passende sedation med …

Representative Results

Tidslinjen og repræsentative resultater er vist i figur 2 og 3. Den kraniel vindue forberedelse, med en lille kranie vindue lateral til den overlegne sagittal sinus (Figur 2 B, C, D) resulterer i en meget lav dødelighed og sygelighed sats, når udføres af en erfaren kirurg. Alle de 10 dyr overlevede, og alle kroniske CW kunne anvendes til høj kvalitet billeddannelse, selv 28 dage efter operationen. Der var ikke noget problem med sår…

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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Cite This Article
Bayerl, S. H., Nieminen-Kelhä, M., Broggini, T., Vajkoczy, P., Prinz, V. Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice. J. Vis. Exp. (118), e54701, doi:10.3791/54701 (2016).

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