We thank all our collaboration partners of the Immunostroke Consortia (FOR 2879, From immune cells to stroke recovery) for suggestions and discussions. This work was funded by the Deutsche Forschungsgemeinschaft (DFG,German Research Foundation) under Germany&#39;s Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy - ID 390857198) and under the grants LI-2534/6-1, LI-2534/7-1 and LL-112/1-1.

The experiments reported in this video were conducted according to the national guidelines for the use of experimental animals, and the protocols were approved by the German governmental committees (Regierung von Oberbayern, Munich, Germany). The mice used in this study were male C57Bl/6J mice, 10-12 weeks old, and dispatched by Charles River Germany. The animals were housed under controlled temperatures (22 &#176;C &#177; 2 &#176;C), with a 12 h light-dark cycle period and access to pelleted food and water ad libitum.
1. Preparation of the material and instruments
<ol>
	<li>Dissolve Rose Bengal in 0.9% saline solution to reach a final concentration of 10 mg/mL. Connect the heat blanket to keep the operation area warm and maintain the mouse body temperature during anesthesia at 37 &#176;C.</li>
	<li>Prepare scissors, forceps, pieces of cotton, dexpanthenol eye ointment, and suture material. Prepare a syringe with saline solution (without needle) to maintain the operation area hydrated. Prepare the anesthesia gas (100% O2 + isoflurane).</li>
</ol>
2. Preparation of the animal
<ol>
	<li>Inject analgesia 30 min before surgery (4 mg/kg Carprofen and 0.1 mg/kg Buprenorphine).</li>
	<li>Record the mouse body weight to adjust the dose of Rose Bengal to be injected (10 &#181;L/g i.e., 100 &#181;g/g).</li>
	<li>Place the mouse in the induction chamber with an isoflurane flow rate of 4% to anesthetize it until the spontaneous movement of the body and vibrissae stops.</li>
	<li>Transfer the mouse into the stereotactic frame and place it in a prone position with its nose into the anesthesia mask. Fix the animal and maintain the isoflurane concentration at 4% for 1 min. Then reduce and maintain the isoflurane concentration at 2%.</li>
	<li>Gently insert the rectal probe to monitor the temperature throughout the surgical procedures. Set the associated feedback-controlled heating pad to maintain the mouse body temperature at 37 &#176;C.</li>
	<li>Apply dexpanthenol eye ointment to both eyes and clean the skin and surrounding fur with a disinfectant agent.</li>
</ol>
3. Photothrombosis model
<ol>
	<li>Make a 2.0-2.5 cm longitudinal incision and retract to expose the skull. Perform the skull exposure with a single cut to avoid wound complications.</li>
	<li>Remove the periosteum gently with cotton and identify the coronal sutures.</li>
	<li>Put on the protective glasses, switch on the 561 nm laser and mark the bregma +3 mm left.</li>
	<li>Switch off the laser, attach a sticker with a 4 mm diameter hole placed at the marked coordinates mentioned above.</li>
	<li>Inject the mouse with Bengal Rose (10 &#181;L/g), intraperitoneally. Place the laser beam at 4-5 cm from the skull, switch on the 561 nm laser and illuminate the skull for 20 min.</li>
	<li>Apply two drops of 0.9% saline on&#160;the skull to rehydrate, suture the wound, and place the animal in a recovery chamber at 37 &#176;C to recover from anesthesia. After 1 h, return the mice to their cages in a temperature-controlled room.</li>
	<li>Inject analgesia every 12 h for 3 days after surgery (4 mg/kg Carprofen and 0.1 mg/kg Buprenorphine).</li>
</ol>
4. Sham operation
<ol>
	<li>Carry out two different procedures of Sham operations as described in steps 4.1.1 and 4.1.2.
	<ol>
		<li>Perform all the procedures identically to the operation described above. Inject Rose Bengal without switching on the laser. After 20 min under anesthesia, allow the animals to stay in the recovery chamber for 1 h to recover, before being returned to their cages.</li>
		<li>Perform all the procedures identically to the operation described above, switching on the laser. Do not inject Rose Bengal. After 20 min of laser illumination, allow the animals to stay in the recovery chamber for 1 h to recover from anesthesia, before being returned to their cages.</li>
	</ol>
	</li>
</ol>
5. Laser speckle
<ol>
	<li>Connect the heated blanket to keep the operation area warm and maintain the mouse body temperature during anesthesia at 37 &#176;C.</li>
	<li>Place the mouse into the induction chamber with an isoflurane flow rate of 4% to anesthetize it until the spontaneous movement of the body and vibrissae stops and then transfer the mouse into the stereotactic frame.</li>
	<li>Place the mouse in a prone position with its nose into the anesthesia mask. Fix the animal and maintain the isoflurane concentration at 4% for 1 min. T hen reduce and maintain it at 2%.</li>
	<li>Gently insert the rectal probe to monitor the temperature throughout the surgical procedures. Set the associated feedback-controlled heating pad to maintain the mouse body temperature at 37 &#176;C and apply dexpanthenol eye ointment to both eyes. Clean the skin and the surrounding fur with a disinfectant agent.</li>
	<li>Make a 2.0-2.5 cm longitudinal incision and retract to expose the skull. Perform the skull exposure with a single cut to avoid wound complications.</li>
	<li>Place the sterotactic frame under the laser speckle and adjust the height to obtain a sharp image. Focus the laser speckle perfusion imaging (LSI) camera on the cranial window. Configure the high resolution laser speckle imaging (LSI) camera system as previously described15.</li>
	<li>Acquire data from a 1 cm x 1 cm field of view using a 785 nm wavelength and 80 mW lasers with a frame rate of 21 images/s at a working distance of 1 cm for 1 min.</li>
	<li>After imaging, apply two drops of 0.9% saline to the skull to rehydrate, suture the wound and place the animal in a recovery chamber at 37 &#176;C to recover from anesthesia for 1 h. After 1 h, return the mice to their cages in a temperature-controlled room.</li>
</ol>
6. Neuroscore
NOTE: For the neurological deficit analysis, a modified neurological scale published by Eckenstein et al. in 1997 is used15.
<ol>
	<li>Score the animals for general (Table 1) and focal deficits (Table 2). This composite scale ranges from 0 (no deficits) to 46 (severe impairments).</li>
	<li>Perform the neuroscore at the same time each day and use surgical clothes to keep a neutral smell.</li>
	<li>Habituate the mice for 30 min in the room with an open cage prior to the testing and allow them to observe each item for 30 s.</li>
</ol>
7. Perfusion
<ol>
	<li>Prepare a 20 mL syringe containing PBS-heparin (2 U/mL) and place it 1 m above the bench to facilitate/ensure gravity-driven perfusion.</li>
	<li>Inject intraperitoneally 100 &#181;L of ketamine and xylazine (120/16 mg/kg body weight, respectively). Wait for 5 min and corroborate the cessation of spontaneous body movement and vibrissae.</li>
	<li>Fix the animal in a supine position and disinfect the abdominal body surface with 100% ethanol. Make a 3 cm long incision in the abdomen; cut the diaphragm and the ribs to completely visualize the heart.</li>
	<li>Make a small incision in the right atrium and insert the perfusion cannula into the left ventricle and perfuse with 20 mL PBS-heparin.</li>
	<li>After perfusion, decapitate the animal and remove the brain, freeze it using dry ice and store them at -80 &#176;C until further use.</li>
</ol>
8. Infarct volumetry
<ol>
	<li>Cryosectioning: Cut the brain serially on a cryostat to 20 &#181;m thick sections every 120 &#181;m and mount on slides. Store the slides at -80 &#176;C until further processing.</li>
	<li>Cresyl violet (CV) staining
	<ol>
		<li>To prepare the staining solution, mix 0.5 g of CV acetate in 500 mL of H2O. Stir and heat (60 &#176;C) until the crystals are dissolved. Allow the solution to cool and store it in a dark bottle. Reheat to 60 &#176;C and filter (paper filter) before every use.</li>
		<li>Dry the slides at room temperature for 30 min. Place them in 95% ethanol for 15 min, followed by 70% ethanol for 1 min, and afterwards in 50% ethanol for 1 min.</li>
		<li>Place the slides in distilled water for 2 min, refresh the distilled water, and place the slides in water again for 1 min. Then, place the slides in the pre-heated staining solution for 10 min at 60 &#176;C. Wash the slides twice in distilled water for 1 min.</li>
		<li>Place the slides in 95% ethanol for 2 min. Then place them into 100% ethanol for 5 min, refresh the 100% ethanol and place the slides in 100% ethanol again for 2 min. Afterwards, cover the slides with a mounting medium.</li>
		<li>Analysis: Scan the slides and analyze the indirect infarct volume by the Swanson method16 to correct for edema: Ischemic area = (ischemic region)-((ipsilateral hemisphere) - (contralateral hemisphere)).</li>
	</ol>
	</li>
</ol>
9. Tunel staining (in situ apoptosis detection kit)
<ol>
	<li>Dry the slides, post-fix in 4% paraformaldehyde in PBS (ph 7.4) for 10-20 min at RT. Wash in PBS, post-fix in precooled ethanol: acetic acid 2:1 for 5 min at -20 &#176;C.</li>
	<li>Wash in PBS and apply equilibration buffer (10 s to a maximum of 60 min at RT) and apply working strength TdT enzyme (1 h at 37 &#176;C in humidified chamber)</li>
	<li>Apply working strength stop/wash enzyme (10 min at RT), wash in PBS and apply warmed (RT) working strength anti-digoxigenin conjugate (30 min at RT in dark)</li>
	<li>Wash in PBS, incubate with DAPI for 5 min at RT and mount the slides with fluoromount media.</li>
</ol>

<ol>
	<li>GBD 2016 Causes of Death Collaborators. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global,+regional,+and+national+age-sex+specific+mortality+for+264+causes+of+death,+1980-2016:+a+systematic+analysis+for+the+Global+Burden+of+Disease+Study+2016.">Global, regional, and national age-sex specific mortality for 264 causes of death, 1980-2016: a systematic analysis for the Global Burden of Disease Study 2016.</a> Lancet. 390 (10100), 1151-1210 (2017).</li><li>Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+focal+stroke:+size,+mechanism,+and+purpose.">Rodent models of focal stroke: size, mechanism, and purpose.</a> NeuroRx: The Journal of the American Society for Experimental Neuro Therapeutics. 2 (3), 396-409 (2005).</li><li>Rosenblum, W. I., El-Sabban, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+aggregation+in+the+cerebral+microcirculation:+effect+of+aspirin+and+other+agents.">Platelet aggregation in the cerebral microcirculation: effect of aspirin and other agents.</a> Circulation Research. 40 (3), 320-328 (1977).</li><li>Watson, B. D., Dietrich, W. D., Busto, R., Wachtel, M. S., Ginsberg, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+reproducible+brain+infarction+by+photochemically+initiated+thrombosis.">Induction of reproducible brain infarction by photochemically initiated thrombosis.</a> Annals of Neurology. 17 (5), 497-504 (1985).</li><li>Bergeron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+photochemical+cortical+lesions+in+rat+brain.">Inducing photochemical cortical lesions in rat brain.</a> Current Protocols in Neuroscience. , (2003).</li><li>Lee, J. 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=Photochemically+induced+cerebral+ischemia+in+a+mouse+model.">Photochemically induced cerebral ischemia in a mouse model.</a> Surgical Neurology. 67 (6), 620-625 (2007).</li><li>Dietrich, W. D., Watson, B. D., Busto, R., Ginsberg, M. D., Bethea, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced+cerebral+infarction.+I.+Early+microvascular+alterations.">Photochemically induced cerebral infarction. I. Early microvascular alterations.</a> Acta Neuropathologica. 72 (4), 315-325 (1987).</li><li>Labat-gest, V., Tomasi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photothrombotic+ischemia:+a+minimally+invasive+and+reproducible+photochemical+cortical+lesion+model+for+mouse+stroke+studies.">Photothrombotic ischemia: a minimally invasive and reproducible photochemical cortical lesion model for mouse stroke studies.</a> Journal of Visualized Experiments: JoVE. (76), e50370 (2013).</li><li>McNutt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Journals+unite+for+reproducibility.">Journals unite for reproducibility.</a> Science. 346 (6210), 679 (2014).</li><li>Llovera, G., 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=Results+of+a+preclinical+randomized+controlled+multicenter+trial+(pRCT):+Anti-CD49d+treatment+for+acute+brain+ischemia.">Results of a preclinical randomized controlled multicenter trial (pRCT): Anti-CD49d treatment for acute brain ischemia.</a> Science Translational Medicine. 7 (299), (2015).</li><li>Dirnagl, U., 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=A+concerted+appeal+for+international+cooperation+in+preclinical+stroke+research.">A concerted appeal for international cooperation in preclinical stroke research.</a> Stroke. 44 (6), 1754-1760 (2013).</li><li>Bath, P. M., Macleod, M. R., Green, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emulating+multicentre+clinical+stroke+trials:+a+new+paradigm+for+studying+novel+interventions+in+experimental+models+of+stroke.">Emulating multicentre clinical stroke trials: a new paradigm for studying novel interventions in experimental models of stroke.</a> International Journal of Stroke: Official Journal of the INternational Stroke Society. 4 (6), 471-479 (2009).</li><li>Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+bioscience+research+reporting:+The+ARRIVE+guidelines+for+reporting+animal+research.">Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research.</a> Journal of Pharmacology &amp; Pharmacotherapeutics. 1 (2), 94-99 (2010).</li><li>Llovera, G., Liesz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+next+step+in+translational+research:+lessons+learned+from+the+first+preclinical+randomized+controlled+trial.">The next step in translational research: lessons learned from the first preclinical randomized controlled trial.</a> Journal of Neurochemistry. 139, 271-279 (2016).</li><li>Gnyawali, S. C., 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=Retooling+laser+speckle+contrast+analysis+algorithm+to+enhance+non-invasive+high+resolution+laser+speckle+functional+imaging+of+cutaneous+microcirculation.">Retooling laser speckle contrast analysis algorithm to enhance non-invasive high resolution laser speckle functional imaging of cutaneous microcirculation.</a> Scientific Reports. 7, 41048 (2017).</li><li>Swanson, G. M., Satariano, E. R., Satariano, W. A., Threatt, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Racial+differences+in+the+early+detection+of+breast+cancer+in+metropolitan+Detroit,+1978+to+1987.">Racial differences in the early detection of breast cancer in metropolitan Detroit, 1978 to 1987.</a> Cancer. 66 (6), 1297-1301 (1990).</li><li>Clark, W. M., Lessov, N. S., Dixon, M. P., Eckenstein, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monofilament+intraluminal+middle+cerebral+artery+occlusion+in+the+mouse.">Monofilament intraluminal middle cerebral artery occlusion in the mouse.</a> Neurological Research. 19 (6), 641-648 (1997).</li><li>Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20 (1), 84-91 (1989).</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> Journal of Visualized Experiments: JoVE. (47), e2423 (2011).</li><li>Tamura, A., Graham, D. I., McCulloch, J., Teasdale, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+cerebral+ischaemia+in+the+rat:+1.+Description+of+technique+and+early+neuropathological+consequences+following+middle+cerebral+artery+occlusion.">Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 1 (1), 53-60 (1981).</li><li>Chen, S. T., Hsu, C. Y., Hogan, E. L., Maricq, H., Balentine, J. 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+model+of+focal+ischemic+stroke+in+the+rat:+reproducible+extensive+cortical+infarction.">A model of focal ischemic stroke in the rat: reproducible extensive cortical infarction.</a> Stroke. 17 (4), 738-743 (1986).</li><li>Tureyen, K., Vemuganti, R., Sailor, K. A., Dempsey, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infarct+volume+quantification+in+mouse+focal+cerebral+ischemia:+a+comparison+of+triphenyltetrazolium+chloride+and+cresyl+violet+staining+techniques.">Infarct volume quantification in mouse focal cerebral ischemia: a comparison of triphenyltetrazolium chloride and cresyl violet staining techniques.</a> Journal of Neuroscience Methods. 139 (2), 203-207 (2004).</li><li>Llovera, G., Roth, S., Plesnila, N., Veltkamp, R., Liesz, A. <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:+permanent+coagulation+of+the+distal+middle+cerebral+artery.">Modeling stroke in mice: permanent coagulation of the distal middle cerebral artery.</a> Journal of Visualized Experiments: JoVE. (89), e511729 (2014).</li><li>Cramer, J. 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=In+vivo+widefield+calcium+imaging+of+the+mouse+cortex+for+analysis+of+network+connectivity+in+health+and+brain+disease.">In vivo widefield calcium imaging of the mouse cortex for analysis of network connectivity in health and brain disease.</a> Neuroimage. 199, 570-584 (2019).</li><li>Heindl, 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=Automated+morphological+analysis+of+microglia+after+stroke.">Automated morphological analysis of microglia after stroke.</a> Frontiers in Cellular Neuroscience. 12, 106 (2018).</li><li>Nih, L. R., Gojgini, S., Carmichael, S. T., Segura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-function+injectable+angiogenic+biomaterial+for+the+repair+of+brain+tissue+following+stroke.">Dual-function injectable angiogenic biomaterial for the repair of brain tissue following stroke.</a> Nature Materials. 17 (7), 642-651 (2018).</li><li>Rust, 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=Nogo-A+targeted+therapy+promotes+vascular+repair+and+functional+recovery+following+stroke.">Nogo-A targeted therapy promotes vascular repair and functional recovery following stroke.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (28), 14270-14279 (2019).</li><li>Kitano, H., Kirsch, J. R., Hurn, P. D., Murphy, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhalational+anesthetics+as+neuroprotectants+or+chemical+preconditioning+agents+in+ischemic+brain.">Inhalational anesthetics as neuroprotectants or chemical preconditioning agents in ischemic brain.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 27 (6), 1108-1128 (2007).</li><li>Farr, T. D., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+qualitative+impairments+in+skilled+reaching+in+the+mouse+(Mus+musculus)+after+a+focal+motor+cortex+stroke.">Quantitative and qualitative impairments in skilled reaching in the mouse (Mus musculus) after a focal motor cortex stroke.</a> Stroke. 33 (7), 1869-1875 (2002).</li><li>Kassem-Moussa, H., Graffagnino, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonocclusion+and+spontaneous+recanalization+rates+in+acute+ischemic+stroke:+a+review+of+cerebral+angiography+studies.">Nonocclusion and spontaneous recanalization rates in acute ischemic stroke: a review of cerebral angiography studies.</a> Archives of Neurology. 59 (12), 1870-1873 (2002).</li><li>Sigler, A., Goroshkov, A., Murphy, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hardware+and+methodology+for+targeting+single+brain+arterioles+for+photothrombotic+stroke+on+an+upright+microscope.">Hardware and methodology for targeting single brain arterioles for photothrombotic stroke on an upright microscope.</a> Journal of Neuroscience Methods. 170 (1), 35-44 (2008).</li></ol>

The authors have no competing interests to disclose.

The presented protocol describes the experimental stroke model of photothrombosis by illuminating the intact skull with a 561 nm laser, with a previous intraperitoneal injection of Rose Bengal. Until recently, the use of this model has been low but is steadily increasing.
Mortality during stroke induction in this model is absent. The overall mortality of less than 5% arises during operation due to anesthesiological complications or sacrifice after meeting the exclusion criteria. To warrant the low variability of this model and its reproducibility, the following exclusion criteria are suggested: 1) operation time longer than 30 min; 2) infection of the suture; 3) bite wound; and 4) no infarct or no fore asymmetry at 24 h after PT.
A widely used experimental stroke models is the transient occlusion of the MCA, by using a suture filament, which is introduced in the internal carotid artery until the silicon-coated tip occludes the origin of the MCA. This model allows the reperfusion by removing the filament and mimics the human clinical scenario, in which there is a restoration of the cerebral blood flow after spontaneous or therapeutic (rtPA) lysis of an embolic clot18,19. However, it involves a complex surgery with high variability of the final infarct and high mortality rate10. In contrast, the permanent occlusion of the MCA distal of the lenticulostriatal arteries can be achieved by coagulation of the artery20,21, which induces locally defined lesions in the neocortex22. Although this model has a lower mortality rate, it requires invasive surgery to the animal by trepanation of the skull over the MCA to later coagulate it23. Consequently, high surgical skills are required for a successful and unbiased in vivo stroke study.
Compared to other brain ischemia models, the photothrombotic model as carried out in this video has the advantage of no craniotomy or major surgery on the animal, unlike other models that involve complex surgeries or brain craniotomy. Moreover, the simple execution of the model makes the surgery accessible to many with low time-consuming training. Low mortality, moderate infarct volume, and flexibility to induce the lesion to a specific brain region, emphasize the advantage of this experimental paradigm for brain regeneration and stroke studies24,25,26,27.
Despite the obvious advantages, a few limitations of this stroke model should be taken into consideration. The long exposure of anesthetics to the animal might be a critical factor to take into account, as the impact of anesthetics on neuroprotection and stroke outcome is already well-known28. Although the duration of this surgical procedure takes approximately 30 min, the animal can be under low anesthetic concentrations due to the minimal manipulation of the animal during the 20 min of laser illumination. Because this model induces moderate brain injuries, only minor behavioral deficits are detectable. Thus, more advanced test systems with higher sensitivity and qualitative test parameters, such as the skilled reaching test29 and Neuroscore17, as described in here, maybe more suitable for detecting long-term functional outcomes in this model. Finally, due to the permanent aggregation of the platelets into the illuminated blood vessels, no reperfusion can be obtained, which is a feature observed in a substantial percentage of stroke patients due to spontaneous clot lysis or therapy30.
A similar phototrombotic stroke model was published in 2013 by Labat-gest and Tomasi, describing a PT protocol using a cold light lamp instead of a 561 nm green laser8. Both laser and cold light sources can be used to induce Rose Bengal excitation. An advantage of laser-based light sources over cold light lamps is that lasers can be used to target individual surface arterioles for in vivo vessel-specific clotting31. Although we were not targeting specific arterioles, we used a 561 nm green laser for brain illuminationn and phototrombosis induction, because of the Rose Bengal absortion peak at 562 nm. To ensure a proper laser intensity during the illumination, the Cobolt Monitor Software-6.1.0.0 was used to calibrate the laser. Moreover, in the present study a Rose Bengal dosage of 10 &#181;L/g (100 &#181;g/g) was sufficient to induce phototrombosis, while the previous protocol reported a higher dose (150 &#181;g/g)8. In addition, the protocol provides a behavioral method to analyze the stroke outcome (Neuroscore) and an additional sham-control group (laser illumination) in order to prove that the laser itself does not produce any tissue damage, so only the combination of Rose Bengal + laser illumination induce a brain lesion.
Overall, this non-invasive straightforward surgical procedure enables high reproducibility and directionality of the stroke lesion to the brain alongside the possibility of long-term observation due to minimal mortality. This photothrombotic stroke model is distinguished as a valuable experimental paradigm for basic and translational stroke research.

Ischemic stroke remains a principal cause of death and acquired adult disability in developed countries in the 21st century accounting for approximately 2.7 million deaths in 2017 worldwide1. Even with the immense efforts of the scientific community, few treatments are available. Furthermore, with such high exclusion criteria, these already limited options are not accessible to many patients, resulting in an urgent need for novel treatments to improve functional recovery after stroke.
Considering the incapability of in vitro models to replicate the complex interactions of the brain, animal models are essential for preclinical stroke research. Mice are the most frequently used animal model in the stroke research field. The majority of these mouse models aim to induce infarctions by blocking the blood flow within the middle cerebral artery (MCA) since the majority of human stroke lesions are located in the MCA territory2. Although these models better recapitulate human stroke lesions, they involve convulated surgeries with high infarct volume variability.
Since Rosenblum and El-Sabban&#39;s proposal of the photothrombotic model in 19773, and later the application of this model to rats Watson et al.4, it has become widely used in ischemic stroke research5,6. The photothrombotic stroke model induces a local and defined cortical infarct as a result of the photoactivation of a light-sensitive dye previously injected into the blood flow. This causes local thrombosis of the vessels in the areas exposed to light. Briefly, upon exposure to light from the injected photosensitive dye, localized oxidative injury of the endothelial cell membrane is induced, leading to platelet aggregation and thrombus formation, followed by local disruption of cerebral blood flow7.
The principal advantage of this technique resides in its simplicity of execution and the possibility to direct the lesion to the desired region. Unlike other experimental stroke models, minor surgical expertise is needed to perform the photothrombotic stroke model as the lesion is induced through illumination of the intact skull. Moreover, the well-delimited borders (Figure 2A and Figure 5B) and the flexibility to induce the lesion to a specific brain region can facilitate the study of cellular responses within the ischemic or intact cortical area8. For these reasons, this approach is suitable for the study of cellular and molecular mechanisms of cortical plasticity.
Over the past few decades, the growing concern regarding the lack of reproducibility between research groups has been coined the so-called replication crisis9. After the coordination of the first preclinical randomized controlled multicenter trial study in 201510, a proposed tool to improve preclinical research11,12,13, it was confirmed that one cause for failing reproducibility between preclinical studies from independent laboratories was the lack of sufficient standardization of experimental stroke models and outcome parameters14. Accordingly, when the ImmunoStroke consortium was established (https://immunostroke.de/), a collaboration which aims to understand brain-immune interactions underlying the mechanistic principles of stroke recovery, the standardization of all the experimental stroke models among each research group was essential.
Described here is the standardized procedure for the induction of the photothrombotic model as used in the above-mentioned research consortium. Briefly, an animal underwent anesthetics, received a Rose Bengal injection (10 &#181;L/g) intraperitonally, and the intact skull, 3 mm left from bregma, was immediately illuminated by a 561 nm laser for 20 min (Figure 1). Additionally, a related histological and behavioral method to analyze the stroke outcome in this model is reported. All methods are based on standard operating procedures developed and used in the laboratory.

<table><tbody><tr><td>561 nm wavelenght laser</td><td>Solna</td><td>Cobolt HS-03</td><td></td></tr><tr><td>Acetic Acid</td><td>Sigma Life Science</td><td>695092</td><td></td></tr><tr><td>Anesthesia system for isoflurane</td><td>Drager</td><td></td><td></td></tr><tr><td>ApopTag Peroxidase In Situ Apoptosis Detection Kit</td><td>Millipore</td><td>S7100</td><td></td></tr><tr><td>Bepanthen pomade</td><td>Bayer</td><td>1578681</td><td></td></tr><tr><td>C57Bl/6J mice</td><td>Charles River</td><td>000664</td><td></td></tr><tr><td>Collimeter</td><td>Thorlabs</td><td>F240APC-A</td><td></td></tr><tr><td>Cotons</td><td>NOBA Verbondmitel Danz</td><td>974116</td><td></td></tr><tr><td>Cresyl violet</td><td>Sigma Life Science</td><td>C5042-10G</td><td></td></tr><tr><td>Cryostat</td><td>Thermo Scientific CryoStarNX70</td><td></td><td></td></tr><tr><td>Ethanol 70%</td><td>CLN Chemikalien Laborbedorf</td><td>521005</td><td></td></tr><tr><td>Ethanol 96%</td><td>CLN Chemikalien Laborbedorf</td><td>522078</td><td></td></tr><tr><td>Ethanol 99%</td><td>CLN Chemikalien Laborbedorf</td><td>ETO-5000-99-1</td><td></td></tr><tr><td>Filter paper</td><td>Macherey-Nagel</td><td>432018</td><td></td></tr><tr><td>Fine Scissors</td><td>FST</td><td>15000-00</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>11616-15</td><td></td></tr><tr><td>Heating blanket</td><td>FHC DC Temperature Controller</td><td>&amp;nbsp;40-90-8D</td><td></td></tr><tr><td>Isoflurane</td><td>Abbot</td><td>B506</td><td></td></tr><tr><td>Isopentane</td><td>Fluka</td><td>59070</td><td></td></tr><tr><td>Ketamine</td><td>Inresa Arzneimittel GmbH</td><td></td><td></td></tr><tr><td>Laser Speckle</td><td>Perimed</td><td>PeriCam PSI HR</td><td></td></tr><tr><td>Mayor Scissors</td><td>FST</td><td>1410-15</td><td></td></tr><tr><td>Phosphate Buffered Saline PH: 7.4</td><td>Apotheke Innestadt Uni Munchen</td><td>P32799</td><td></td></tr><tr><td>Protective glasses</td><td>Laser 2000</td><td>NIR-ZS2-38</td><td></td></tr><tr><td>Rose Bengal</td><td>Sigma Aldrich</td><td>198250-5G</td><td></td></tr><tr><td>Roti-Histokit mounting medium</td><td>Roth</td><td>6638.1</td><td></td></tr><tr><td>Saline solution</td><td>Braun</td><td>131321</td><td></td></tr><tr><td>Stereomikroskop</td><td>Zeiss</td><td>Stemi DV4</td><td></td></tr><tr><td>Stereotactic frame</td><td>Stoelting</td><td>51500U</td><td></td></tr><tr><td>Superfrost Plus Slides</td><td>Thermo Scientific</td><td>J1800AMNZ</td><td></td></tr><tr><td>Xylacine</td><td>Albrecht</td><td></td><td></td></tr></tbody></table>

modeling stroke in mice: focal cortical lesions by photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

The model described here is a photothrombotic stroke model by Rose Bengal injection and intact skull illumination for 20 min, at a constant 561 nm wavelength and 25 mW output power at the fiber. Although the complete photothrombotic surgery lasts 30 min, the animal is kept under low anesthesia and the brain damage is moderate. Approximately 10 min after transfer to their cages, all the animals were awake, freely moving in the cage, and interacting with littermates.
Infarct volumetry was performed using cresyl violet stained serial coronal brain sections 24 h after stroke induction (Figure 2A). The mean infarct volume was 29.3 mm3, representing 23% of one brain hemisphere. Moreover, the variability of this stroke model is exceptionally low with a standard deviation of approximately 3.5% (Figure 2B). The lesion area encompasses the motor cortex without the affection of subcortical structures.
Photothrombosis caused a moderate, long-term sensorimotor impairment, indicated by the composite Neuroscore17 (Figure 3); general and focal deficits were measured 24 h, 3 days and 7 days after surgery. The general Neuroscore has five items, including the evaluation of the fur, ears, eyes, posture, and spontaneous activity, with a maximum score of 18 (Table 1). The focal Neuroscore comprises seven items, including the evaluation of body symmetry, gait, climbing, circling behavior, forelimb symmetry, compulsory cycling, and whiskers response, with a maximum score of 28 (Table 2). Stroke animals had a significant change in the composite neuroscore 24 h after surgery compared to Sham-operated animals. These differences persisted, although stroke mice improved over time (Figure 3).
Mortality during the observation time rarely occurs in 1-2% of the animals. In this report, none of the 10 animals studied had to be excluded and all of them survived the 7-day observation period. The body weight and temperature changes in the mice were monitored at 24 h, 3 days, and 7 days after surgery (Figure 4A,B). Data showed that body weight and temperature were decreased 24 h after surgery only in the Rose Bengal + illumination group, but recovered to the level of the Sham-operated animals in 3 days after surgery.
To confirm an induction of ischemic changes, 24 h after surgery, the animals underwent a laser imaging test. A laser speckle contrast imaging measured blood perfusion of the cortex for a duration of 1 min and an averaged color-coded picture was obtained for each animal. This demonstrates&#160;that Rose Bengal or laser illumination alone does not produce a lesion, while simultaneous application of Rose Bengal and laser illumination generates a round hypoperfused area of 4 mm diameter surrounded by a narrow oligemic zone (Figure 5A). In addition, a cresyl violet and Tunel staining for assessment of the infarct volume 24 h after surgery revealed no tissue damage either in Rose Bengal or laser illumination surgeries. On the other hand, Rose Bengal + laser illumination generated a well-demarcated lesion (Figure 5B).
Table 1: General Neuroscore. For each of the five general deficits measured, animals can receive between 0 and 4 points depending on the severity. The scores on the five areas are then summed to provide a total general score ranging from 0-18. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62536/Table 1-62536R1.pdf" target="_blank">Please click here to download this Table.</a>
Table 2: Focal Neuroscore. For each of the seven general deficits measured, animals can receive between 0 and 4 points depending on the severity. The scores on the five areas are then summed to provide a total general score ranging from 0-28. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62536/Table 2- 62536R1.pdf" target="_blank">Please click here to download this Table.</a>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62536/62536fig01.jpg" /> 
Figure 1: Photothrombosis (PT). Diagram depicting the photothrombotic area, 3 mm from Bregma. The green dot indicates the position of the laser. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62536/62536fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62536/62536fig02.jpg" /> 
Figure 2: Volumetric infarct analysis and infarct outcome 24 h after PT. (A) Representative cresyl violet stained coronal brain, sections every 120 &#181;m at 24 h after PT. Dashed lines demarcate the lesion area. (B) Infarct volume analysis of 10 brains (each dot representing one individual brain) 24 h after PT. The horizontal red line represents the mean (29.32 mm3), error bars indicate standard deviation (3.45 mm3). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62536/62536fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62536/62536fig03.jpg" /> 
Figure 3: Neuroscore for functional deficits after PT. Composite Neuroscore before, 24 h, 3 days, and 7 days after PT. BL = before PT, RB = Rose Bengal. n = 5 per group. *p &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62536/62536fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62536/62536fig04.jpg" /> 
Figure 4: Body weight and temperature analysis after PT. (A) Body weight and (B) temperature was slightly reduced in PT animals compared to Sham-operated groups at 24 h and recovered 3 days after PT. BL = before PT, RB = Rose Bengal. n = 5 per group.*p &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62536/62536fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62536/62536fig05.jpg" /> 
Figure 5: Lesion confirmation after PT. (A) Laser Speckle imaging (B) Cresyl violet (upper panels) and Tunel staining (lower panels) confirmed the lesion only after administration of Rose Bengal and subsequent laser illumination. RB = Rose Bengal. Scale bar = 1,000 &#181;m in upper panel B, scale bar = 20 &#181;m in lower panel B. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62536/62536fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis at JoVE.com

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

modeling-stroke-in-mice-focal-cortical-lesions-by-photothrombosis

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.1K Views.  LMU Munich. De fototrombose beroerte model benadering kan door geschikt voor cellulaire en moleculaire studies van corticale plasticiteit vanwege de beperkte grenzen en flexibiliteit om letsel te induceren in verschillende gebieden van de cortex. Drie belangrijkste voordelen onderscheiden het fototrombosemodel van andere beroertemodellen, de mogelijkheid om de laesie naar het gewenste gebied te leiden, het is hoge reproduceerbaarheid en lage mortaliteit. Een goede rose bengalen dosering is van cruciaal b...

Video: Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Medicine

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires &#34;cryofixation&#34; of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 &#176;C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at &#8804;-160 &#176;C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 &#176;C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their &#34;characteristic&#34; peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

This paper describes the application of cryoanalytical electron microscopy to the quantitative measurement of total calcium content and distribution at subcellular resolution in physiologically defined biological specimens.

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for white matter stroke has hampered research investigations. Here, we describe a novel method for creating a circumscribed capsular infarct that minimizes damage to neighboring gray matter structures. We used pre-surgery neural tracing with adeno-associated virus-green fluorescent protein (AAV-GFP) to identify somatotopic organization of the forelimb area within the internal capsule. The adjustment of light intensity based on different optical properties of gray and white matter contributes to selective destruction of white matter with relative preservation of gray matter. Accurate positioning of optical-neural interface enables destruction of entire forelimb area in the internal capsule, which leads to a marked and persistent motor deficit. Thus, this technique produces highly replicable capsular infarct lesions with a persistent motor deficit. The model will be helpful not only to study white matter stroke (WMS) at the behavioral, circuit, and cellular levels, but also to assess its usefulness for development of new therapeutic and rehabilitative interventions.

This manuscript describes a modeling technique of capsular infarct. Here we utilized a modified photothrombotic technique with low intensity of light after pre-surgery target mapping. Using this technique, we created a circumscribed capsular infarct model with persistent motor impairment.

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for ...

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and contributes significantly to the development of vascular dementia. Few models of focal white matter stroke exist and this lack of appropriate models has hampered understanding of the neurobiologic mechanisms involved in injury response and repair after this type of stroke. The main limitation of other subcortical stroke models is that they do not focally restrict the infarct to the white matter or have primarily been validated in non-murine species. This limits the ability to apply the wide variety of murine research tools to study the neurobiology of white matter stroke. Here we present a methodology for the reliable production of a focal stroke in murine white matter using a local injection of an irreversible eNOS inhibitor. We also present several variations on the general protocol including two unique stereotactic variations, retrograde neuronal tracing, as well as fresh tissue labeling and dissection that greatly expand the potential applications of this technique. These variations allow for multiple approaches to analyze the neurobiologic effects of this common and understudied form of stroke.

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

Modeling Photothrombotic Stroke in Mouse Model: A Laser Illumination Technique Through Mouse Skull Following Photosensitive Dye Administration to Induce Photothrombosis

Source: Llovera, G. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62536">Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis.</a> J. Vis. Exp. (2021)
This video demonstrates the procedure to generate photothrombotic stroke in a mouse model by laser illumination of an intact skull following administration of photosensitive dye.

Source: Llovera, G. et al. Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis. J. Vis. Exp. (2021)
This video demonstrates the procedure ...

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

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