VP is a participant in the Charit&#233; Clinical Scientist Program, funded by the Charit&#233; - Universit&#228;tsmedizin 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.

ETHICS STATEMENT: Experiments involving animal subjects were performed in accordance with the guidelines and regulations set forth by Landesamt fuer Gesundheit und Soziales, Berlin, Germany (G0298/13) and the ARRIVE criteria, as applicable. For this study, 10- to 12-week-old male C57Bl/6J mice were used.
1. Lateral Chronic Cranial Window Preparation
<ol>
	<li>Perform anesthesia with a subcutaneous injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). Test for adequate sedation with a pai...

<ol>
	<li>Mukherjee, D., Patil, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+the+global+burden+of+stroke.">Epidemiology and the global burden of stroke.</a> World Neurosurg. 76 (6), 85-90 (2011).</li><li>Ebinger, M., Pr&#252;ss, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+use+of+ambulance-based+thrombolysis+on+time+to+thrombolysis+in+acute+ischemic+stroke:+a+randomized+clinical+trial.">Effect of the use of ambulance-based thrombolysis on time to thrombolysis in acute ischemic stroke: a randomized clinical trial.</a> JAMA. 311 (16), 1622-1631 (2014).</li><li>Ebinger, M., Lindenlaub, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prehospital+thrombolysis:+a+manual+from+Berlin.">Prehospital thrombolysis: a manual from Berlin.</a> J vis Exp. (81), e50534 (2013).</li><li>Bogousslavsky, J., Van Melle, G., Regli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lausanne+Stroke+Registry:+analysis+of+1,000+consecutive+patients+with+first+stroke.">The Lausanne Stroke Registry: analysis of 1,000 consecutive patients with first stroke.</a> Stroke. 19 (9), 1083-1092 (1988).</li><li>Engel, O., Kolodziej, S., Dirnagl, U., Prinz, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+stroke+in+mice+-+middle+cerebral+artery+occlusion+with+the+filament+model.">Modeling stroke in mice - middle cerebral artery occlusion with the filament model.</a> J Vis Exp. (47), (2011).</li><li>Donnan, G. A., Fisher, M., Macleod, M., Davis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroke.">Stroke.</a> Lancet. 371 (9624), 1612-1623 (2008).</li><li>Meairs, S., Wahlgren, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroke+research+priorities+for+the+next+decade--A+representative+view+of+the+European+scientific+community.">Stroke research priorities for the next decade--A representative view of the European scientific community.</a> Cerebrovasc Dis. 22 (2-3), 75-82 (2006).</li><li>Rosamond, W., Flegal, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics--2007+update:+a+report+from+the+American+Heart+Association+Statistics+Committee+and+Stroke+Statistics+Subcommittee.">Heart disease and stroke statistics--2007 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee.</a> Circulation. 115 (5), 69-171 (2007).</li><li>Moskowitz, M. A., Lo, E. H., Iadecola, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+science+of+stroke:+mechanisms+in+search+of+treatments.">The science of stroke: mechanisms in search of treatments.</a> Neuron. 67 (2), 181-198 (2010).</li><li>Dirnagl, U., Iadecola, C., Moskowitz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathobiology+of+ischaemic+stroke:+an+integrated+view.">Pathobiology of ischaemic stroke: an integrated view.</a> Trends Neurosci. 22 (9), 391-397 (1999).</li><li>Dirnagl, U., Endres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Found+in+Translation:+Preclinical+Stroke+Research+Predicts+Human+Pathophysiology,+Clinical+Phenotypes,+and+Therapeutic+Outcomes.">Found in Translation: Preclinical Stroke Research Predicts Human Pathophysiology, Clinical Phenotypes, and Therapeutic Outcomes.</a> Stroke. , (2014).</li><li>Prinz, V., Hetzer, A. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+heralds+secondary+nigral+lesion+after+brain+ischemia+in+mice:+a+secondary+time+window+for+neuroprotection.">MRI heralds secondary nigral lesion after brain ischemia in mice: a secondary time window for neuroprotection.</a> J Cereb Blood Flow Metab. , (2015).</li><li>Shih, A. Y., Mateo, C., Drew, P. J., Tsai, P. S., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polished+and+reinforced+thinned-skull+window+for+long-term+imaging+of+the+mouse+brain.">A polished and reinforced thinned-skull window for long-term imaging of the mouse brain.</a> J Vis Exp. (61), (2012).</li><li>Holtmaat, A., Bonhoeffer, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term,+high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nat Protoc. 4 (8), 1128-1144 (2009).</li><li>Iadecola, C., Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microcircualtion--fantastic+voyage:+introduction.">The microcircualtion--fantastic voyage: introduction.</a> Stroke. 44 (6), 83 (2013).</li><li>Blinder, P., Tsai, P. S., Kaufhold, J. P., Knutsen, P. M., Suhl, H., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cortical+angiome:+an+interconnected+vascular+network+with+noncolumnar+patterns+of+blood+flow.">The cortical angiome: an interconnected vascular network with noncolumnar patterns of blood flow.</a> Nat Neurosc. 16 (7), 889-897 (2013).</li><li>Shih, A. Y., Driscoll, J. D., Drew, P. J., Nishimura, N., Schaffer, C. B., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+microscopy+as+a+tool+to+study+blood+flow+and+neurovascular+coupling+in+the+rodent+brain.">Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain.</a> J Cereb Blood Flow Metab. 32 (7), 1277-1309 (2012).</li><li>Cabrales, P., Carvalho, L. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+the+mouse+brain+microcirculation+using+a+closed+cranial+window.">Intravital microscopy of the mouse brain microcirculation using a closed cranial window.</a> J Vis Exp. (45), (2010).</li><li>Rosell, A., Agin, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distal+occlusion+of+the+middle+cerebral+artery+in+mice:+are+we+ready+to+assess+long-term+functional+outcome.">Distal occlusion of the middle cerebral artery in mice: are we ready to assess long-term functional outcome.</a> Transl Stroke Res. 4 (3), 297-307 (2013).</li><li>Dorand, R. D., Barkauskas, D. S., Evans, T. A., Petrosiute, A., Huang, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+intravital+thinned+skull+and+cranial+window+approaches+to+study+CNS+immunobiology+in+the+mouse+cortex.">Comparison of intravital thinned skull and cranial window approaches to study CNS immunobiology in the mouse cortex.</a> Intravital. 3 (2), (2014).</li><li>Balkaya, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+post-stroke+behavior+in+mouse+models+of+focal+ischemia.">Assessing post-stroke behavior in mouse models of focal ischemia.</a> J Cereb Blood Flow Metab. 33 (3), 330-338 (2013).</li><li>Balkaya, M., Kr&#246;ber, J., Gertz, K., Peruzzaro, S., Endres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+long-term+functional+outcome+in+a+murine+model+of+mild+brain+ischemia.">Characterization of long-term functional outcome in a murine model of mild brain ischemia.</a> J Neurosci Methods. 213 (2), 179-187 (2013).</li><li>Freret, T., Bouet, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+deficits+after+distal+focal+cerebral+ischemia+in+mice:+Usefulness+of+adhesive+removal+test.">Behavioral deficits after distal focal cerebral ischemia in mice: Usefulness of adhesive removal test.</a> Beh Neurosci. 123 (1), 224-230 (2009).</li><li>Liu, S., Zhen, G., Meloni, B. P., Campbell, K., Winn, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RODENT+STROKE+MODEL+GUIDELINES+FOR+PRECLINICAL+STROKE+TRIALS+(1ST+EDITION).">RODENT STROKE MODEL GUIDELINES FOR PRECLINICAL STROKE TRIALS (1ST EDITION).</a> J Exp Stroke Trans Med. 2 (2), 2-27 (2009).</li><li>Florian, B., Vintilescu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+hypothermia+reduces+infarct+volume+in+aged+rats+after+focal+ischemia.">Long-term hypothermia reduces infarct volume in aged rats after focal ischemia.</a> Neurosci Lett. 438 (2), 180-185 (2008).</li><li>Noor, R., Wang, C. X., Shuaib, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hyperthermia+on+infarct+volume+in+focal+embolic+model+of+cerebral+ischemia+in+rats.">Effects of hyperthermia on infarct volume in focal embolic model of cerebral ischemia in rats.</a> Neurosci Lett. 349 (2), 130-132 (2003).</li><li>Barber, P. A., Hoyte, L., Colbourne, F., Buchan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-regulated+model+of+focal+ischemia+in+the+mouse:+a+study+with+histopathological+and+behavioral+outcomes.">Temperature-regulated model of focal ischemia in the mouse: a study with histopathological and behavioral outcomes.</a> Stroke. 35 (7), 1720-1725 (2004).</li><li>Shin, H. K., Nishimura, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mild+induced+hypertension+improves+blood+flow+and+oxygen+metabolism+in+transient+focal+cerebral+ischemia.">Mild induced hypertension improves blood flow and oxygen metabolism in transient focal cerebral ischemia.</a> Stroke. 39 (5), 1548-1555 (2008).</li><li>Kapinya, K. J., Prass, K., Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+induced+prolonged+protection+against+cerebral+ischemia+in+mice:+a+redox+sensitive+mechanism.">Isoflurane induced prolonged protection against cerebral ischemia in mice: a redox sensitive mechanism.</a> Neuroreport. 13 (11), 1431-1435 (2002).</li><li>Gertz, K., Priller, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+activity+improves+long-term+stroke+outcome+via+endothelial+nitric+oxide+synthase-dependent+augmentation+of+neovascularization+and+cerebral+blood+flow.">Physical activity improves long-term stroke outcome via endothelial nitric oxide synthase-dependent augmentation of neovascularization and cerebral blood flow.</a> Circ Res. 99 (10), 1132-1140 (2006).</li><li>Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bench+to+bedside:+the+quest+for+quality+in+experimental+stroke+research.">Bench to bedside: the quest for quality in experimental stroke research.</a> J Cereb Blood Flow Metab. 26 (12), 1465-1478 (2006).</li></ol>

Stroke is among the principal causes of long-term disability and death worldwide1. Beyond acute treatment, investigating new approaches and mechanisms to accelerate and enhance recovery after stroke remains a prime medical goal7. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. In fact, models inducing transient or permanent MCAo mimic one of the most common types of focal cerebral ischemia in patients4. Besides proximal occlusion of the MCA, the...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Binocular surgical microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000 C</td>
			<td></td>
		</tr>
		<tr>
			<td>Light source for microscope</td>
			<td>Zeiss</td>
			<td>CL 6000 LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad with rectal probe</td>
			<td>FST</td>
			<td>21061-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic frame</td>
			<td>Kopf</td>
			<td>Model 930</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaethesia system for isoflurane</td>
			<td>Draeger</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abott</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont forceps #5</td>
			<td>FST</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont forceps #7</td>
			<td>FST</td>
			<td>11271-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar Forceps</td>
			<td>Erbe</td>
			<td>20195-501</td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar Forceps&#160;</td>
			<td colspan="2">Erbe&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 20195-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdrill</td>
			<td colspan="2">FST&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 18000-17&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>FST</td>
			<td>12010-14</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 silk suture</td>
			<td>Feuerstein, Suprama</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7-0 silk suture</td>
			<td>Feuerstein,Suprama</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 silk suture</td>
			<td>Feuerstein, Suprama</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Veterinary Recovery Chamber</td>
			<td>Peco Services</td>
			<td>V1200</td>
			<td></td>
		</tr>
	</tbody>
</table>

lateral chronic cranial window preparation enables in vivo observation following distal middle cerebral artery occlusion in mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

The timeline and representative results are shown in Figures 2 and 3. The cranial window preparation, with a small cranial window lateral to the superior sagittal sinus (Figure 2 B, C, D) results in a very low mortality and morbidity rate when performed by an experienced surgeon. All of the 10 animals survived, and all chronic CW could be used for high-quality imaging, even 28 days after surgery. There was no problem with wound infections...

Watch this Scientific Journal Video about Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice at JoVE.com

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

The overall goal of this procedure is the preparation of a lateral cranial window combined with permanent middle cerebral artery occlusion. To facilitate longitudinal intravital microscopy in mice. This method can help to investigate and answer key questions in the field of cerebellar ischemia research.The main advantage of this technique is that it offers in vivo imaging of acute and chronic changes after stroke. Begin by administering anesthesia according to an approved animal protocol. Ensure that a surgical plane of anesthesia has been reached by administering a pain stimulus, such as a toe pinch, and noting the absence of a reaction.Next, use a rodent shaver to remove the fur from the neck to the eyes. Fix the head of the mouse in a Stereotaxic frame. Then cover the eyes with ophthalmic ointment to prevent dehydration.Clean the shaved area to remove all hairs, and sterilize the scalp with three applications of 74.1%ethanol and 10%tube Propranolol. Using a sterile scalpel, make a midline incision from the neck to the eyes. Then span the skin flap with four tenting sutures.Next, carefully scrape the periosteum on the left side of the skull. And remove it to the point where the temporal muscle begins. Now use a micro drill to thin the skull at the rim of the bone flap.To create a four-millimeter diameter frontoparietal craniotomy. Apply saline solution while drilling to avoid heat injury. Once drilled, elevate the bone flap with cannulas and remove it using micro forceps.Irrigate the area carefully and extensively with saline solution. Mix the dental glue until it has the proper consistency. Then apply it to the bone around the craniotomy.Place a six-millimeter diameter cover glass on the prepared glue. And fix it with the rest of the dental glue. Irrigation accelerates the curing process and ensures the seal is water-tight.Probe the dental glue with forceps to ensure that it is dried and hard. After the glue has cured, remove the tenting sutures and close the wound with skin sutures. Administer 0.5 milliliters of sterile saline subcutaneously for fluid balance.After surgery, monitor the animal in a heated recovery cage until recovered from anesthesia. Ensure that the animal is completely recovered before returning it to a cage with other animals. Always administer analgesia after surgery.Here, 10 milligrams per milliliter acetaminophen is added to the drinking water. Always provide mashed animal food in a petri dish placed on the floor to make eating simple and to avoid critical weight loss after surgery. Intravital microscopy can be performed on the first day after cranial window preparation.Apply isoflurane anesthesia according to an approved protocol. Fix the animal in a head-holder and ensure the nose cone is properly fitted. Open the skin suture and clean the window with cotton buds and sterile saline.After 24 hours, the cranial window should be filled with cerebrospinal fluid, which enables imaging. Perform the middle cerebral artery occlusion procedure five days after cranial window preparation, when the immune response to the procedure has receded. Here, an overview after craniotomy for MCAO induction is shown.The intraoperative photo shows the vessels after first and second bipolar contact. The distal MCA appears completely occluded. This picture provides a final overview using lower magnification.After inducing surgical anesthesia with isoflurane as before, shave the fur in the area between the eye and ear, apply ophthalmic ointment to both eyes, and disinfect the skin and surrounding fur with alcohol solution, as before. Place the mouse on a feedback-controlled heating pad to maintain body temperature. Place the animal in the lateral position and fix the nose in the anesthesia mask.Gently separate the skin of the incision made for the cranial window preparation and identify the temporal muscle underneath. Next, adjust the energy of the high frequency generator and use the bipolar mode. Use electrocoagulation forceps to carefully lift the temporal muscle from the skull, creating a muscle flap without totally removing the muscle.Then identify the middle cerebral artery below the transparent skull in the rostral part of the temporal area. Dorsal to the retro-orbital sinus. Thin the skull above the MCA branch with a micro drill, while continuously irrigating to avoid heat damage.Then lift the bone and remove it with micro forceps. Decrease the energy of the high frequency generator to three to five watt. Then, approach the artery from above and gently touch it with the bipolar forceps on both sides without lifting the vessel.Wait for thirty seconds, and then touch the artery gently with the tip of the bipolar forceps to ensure that the blood flow is permanently interrupted. After MCA occlusion, fix the temporal muscle with one or two stitches at the muscle insertion to cover the bone defect. Then, suture the wound and place the animal into the heated cage until recovered.This MRI was performed 24 hours after sham surgery, and does not show any lesioned cortical tissue. Whereas this MRI, performed 24 hours after ischemia, clearly depicts the ischemic lesion indicated by an asterisk, located underneath the chronic cranial window. This image shows intravital epifluorescense imaging and two-photon imaging of the cortical vasculature to monitor changes in microcirculation.Infarct volume assessed via MRI at 24 and 96 hours after ischemia, shows a mean lesion volume of 13.16 millimeters cubed at 24 hours, and 12.2 millimeters cubed at 96 hours. Once mastered, the cranial window preparation can be done in about 30 minutes. And the MCAO in about 15 minutes.Following this procedure, other methods, like MRI, or dual-photon microscopy can be performed to answer additional questions. These new techniques allow investigators to investigate a neurovascular unit at a molecular and cellular level. And most particularly, in the living brain.

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Research

JoVE Journal

Neuroscience

11.5K Görüntüleme.  Charité-Universitätsmedizin. The overall goal of this procedure is the preparation of a lateral cranial window combined with permanent middle cerebral artery occlusion. To facilitate longitudinal intravital microscopy in mice. This method can help to investigate and answer key questions in the field of cerebellar ischemia research.The main advantage of this technique is that it offers in vivo imaging of acute and chronic changes after stroke. Begin by administering anesthesia according to an approved animal protoc...