We would like to thank Nikolaos Plakopitis and Ioannis Tatsidis for valuable surgical support during parts of the study.

<ol>
	<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>Lourbopoulos, A., 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=Translational+block+in+stroke:+A+constructive+and+&amp;#34;out-of-the-box&amp;#34;+reappraisal.">Translational block in stroke: A constructive and &amp;#34;out-of-the-box&amp;#34; reappraisal.</a> Front Neurosci. 15, 652403 (2021).</li><li>Cai, 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=Panoptic+imaging+of+transparent+mice+reveals+whole-body+neuronal+projections+and+skull-meninges+connections.">Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections.</a> Nat Neurosci. 22 (2), 317-327 (2019).</li><li>Fluri, F., Schuhmann, M. 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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=Guidelines+for+the+early+management+of+patients+with+acute+ischemic+stroke:+2019+update+to+the+2018+guidelines+for+the+early+management+of+acute+ischemic+stroke:+A+guideline+for+healthcare+professionals+from+the+american+heart+association/american+stroke+association.">Guidelines for the early management of patients with acute ischemic stroke: 2019 update to the 2018 guidelines for the early management of acute ischemic stroke: A guideline for healthcare professionals from the american heart association/american stroke association.</a> Stroke. 50 (12), e344-e418 (2019).</li><li>Schaar, K. L., Brenneman, M. M., Savitz, S. 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B., 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=Rat+middle+cerebral+artery+occlusion:+Evaluation+of+the+model+and+development+of+a+neurologic+examination.">Rat middle cerebral artery occlusion: Evaluation of the model and development of a neurologic examination.</a> Stroke. 17 (3), 472-476 (1986).</li><li>Bieber, 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=Validity+and+reliability+of+neurological+scores+in+mice+exposed+to+middle+cerebral+artery+occlusion.">Validity and reliability of neurological scores in mice exposed to middle cerebral artery occlusion.</a> Stroke. 50 (10), 2875-2882 (2019).</li><li>Lourbopoulos, A., Karacostas, D., Artemis, N., Milonas, I., Grigoriadis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+a+new+modified+intraluminal+suture+for+temporary+middle+cerebral+artery+occlusion+in+rats+of+various+weight.">Effectiveness of a new modified intraluminal suture for temporary middle cerebral artery occlusion in rats of various weight.</a> J Neurosci Methods. 173 (2), 225-234 (2008).</li><li>Lourbopoulos, A., 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=Inadequate+food+and+water+intake+determine+mortality+following+stroke+in+mice.">Inadequate food and water intake determine mortality following stroke in mice.</a> J Cereb Blood Flow Metab. 37 (6), 2084-2097 (2017).</li><li>De Simoni, M. 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(89), e51729 (2014).</li><li>Dzirkale, Z., 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+behavioural+alterations+in+mice+following+transient+cerebral+ischemia.">Long-term behavioural alterations in mice following transient cerebral ischemia.</a> Behav Brain Res. 452, 114589 (2023).</li><li>Kolabas, Z. I., 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=Distinct+molecular+profiles+of+skull+bone+marrow+in+health+and+neurological+disorders.">Distinct molecular profiles of skull bone marrow in health and neurological disorders.</a> Cell. 186 (17), 3706-3725.e29 (2023).</li><li>Kestner, R. I., 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=Gene+expression+dynamics+at+the+neurovascular+unit+during+early+regeneration+after+cerebral+ischemia/reperfusion+injury+in+mice.">Gene expression dynamics at the neurovascular unit during early regeneration after cerebral ischemia/reperfusion injury in mice.</a> Front Neurosci. 14, 280 (2020).</li><li>Pinto, 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=Bridging+the+transient+intraluminal+stroke+preclinical+model+to+clinical+practice:+From+improved+surgical+procedures+to+a+workflow+of+functional+tests.">Bridging the transient intraluminal stroke preclinical model to clinical practice: From improved surgical procedures to a workflow of functional tests.</a> Front Neurol. 13, 846735 (2022).</li><li>Damigos, 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=Machine+learning+based+analysis+of+stroke+lesions+on+mouse+tissue+sections.">Machine learning based analysis of stroke lesions on mouse tissue sections.</a> J Cereb Blood Flow Metab. 42 (8), 1463-1477 (2022).</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. 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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=Housing-temperature+reveals+energy+intake+counter-balances+energy+expenditure+in+normal-weight,+but+not+diet-induced+obese,+male+mice.">Housing-temperature reveals energy intake counter-balances energy expenditure in normal-weight, but not diet-induced obese, male mice.</a> Commun Biol. 5 (1), 946 (2022).</li><li>Tashiro, M., Hosokawa, Y., Amao, H., Tohei, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duration+of+thermal+support+for+preventing+hypothermia+induced+by+anesthesia+with+medetomidine-midazolam-butorphanol+in+mice.">Duration of thermal support for preventing hypothermia induced by anesthesia with medetomidine-midazolam-butorphanol in mice.</a> J Vet Med Sci. 82 (12), 1757-1762 (2020).</li><li>Taylor, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+two+devices+used+to+maintain+normothermia+in+rats+and+mice+during+general+anesthesia.">Study of two devices used to maintain normothermia in rats and mice during general anesthesia.</a> J Am Assoc Lab Anim Sci. 46 (5), 37-41 (2007).</li><li>Bendele, A. M., Jones, T. C., Hard, G. C., Mohr, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Urinary system. , 456-462 (1998).</li><li>Schallert, T., Fleming, S. M., Leasure, J. L., Tillerson, J. L., Bland, 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=Cns+plasticity+and+assessment+of+forelimb+sensorimotor+outcome+in+unilateral+rat+models+of+stroke,+cortical+ablation,+parkinsonism+and+spinal+cord+injury.">Cns plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury.</a> Neuropharmacology. 39 (5), 777-787 (2000).</li><li>Mathis, A., 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=Deeplabcut:+Markerless+pose+estimation+of+user-defined+body+parts+with+deep+learning.">Deeplabcut: Markerless pose estimation of user-defined body parts with deep learning.</a> Nat Neurosci. 21 (9), 1281-1289 (2018).</li><li>Fonarow, G. 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=Relationship+of+national+institutes+of+health+stroke+scale+to+30-day+mortality+in+medicare+beneficiaries+with+acute+ischemic+stroke.">Relationship of national institutes of health stroke scale to 30-day mortality in medicare beneficiaries with acute ischemic stroke.</a> J Am Heart Assoc. 1 (1), 42-50 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allen+reference+atlas+-+adult+mouse+brain+coronal.">Allen reference atlas - adult mouse brain coronal.</a> Available from atlas.Brain-map.Org. , (2011).</li><li>Groger, 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=Release+of+bradykinin+and+expression+of+kinin+b2+receptors+in+the+brain:+Role+for+cell+death+and+brain+edema+formation+after+focal+cerebral+ischemia+in+mice.">Release of bradykinin and expression of kinin b2 receptors in the brain: Role for cell death and brain edema formation after focal cerebral ischemia in mice.</a> J Cereb Blood Flow Metab. 25 (8), 978-989 (2005).</li><li>Kolbinger, A., 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=Behind+the+wall-compartment-specific+neovascularisation+during+post-stroke+recovery+in+mice.">Behind the wall-compartment-specific neovascularisation during post-stroke recovery in mice.</a> Cells. 11 (10), (2022).</li><li>Merino-Serrais, P., 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=Microanatomical+study+of+pyramidal+neurons+in+the+contralesional+somatosensory+cortex+after+experimental+ischemic+stroke.">Microanatomical study of pyramidal neurons in the contralesional somatosensory cortex after experimental ischemic stroke.</a> Cereb Cortex. 33 (4), 1074-1089 (2023).</li><li>Metz, G. 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No disclosures to report.

The present protocol is a comprehensive guide to the mSU support protocol, designed to reduce artifactual mortality between days 3-10 in mice subjected to large territorial strokes by the fMCAo model13. Additionally, the present protocol includes a standardized video guide for the focal component of the ESS scale (fESS) to reduce the extended subjectivity and bias of neurological focal scoring in mice after stroke. Alongside, we introduce the Ladder-rung test for quantifying long-term (up to 6 mon...

Large ischemic stroke is one of the major causes of morbidity and disability worldwide1. Still, mouse stroke research faces a translational block in almost all preclinically tested treatments so far2. The multiple reasons for this problem and the efforts to overcome it have been reviewed in detail in previous publication2. These challenges arise from the limited integration of clinical advances into relevant animal models2 and the weaknesses of behavioral and neurological scoring systems in detecting post-stroke deficits accurately and sensitively2.
Recent discoveries3 support that the filament model of middle artery occlusion (fMCAo) in mice has significant translational advantages over other models with open skull injury and trauma2,4. On top of that, this is the only one that models the reperfusion and mechanical thrombectomy of clinical stroke settings5,6,7. However, the model faces high subacute mortality between 3-7 days post-stroke, which prohibits long-term studies of large strokes and was considered until recently the model&#180;s inherent and insurmountable artifact. In addition, the detection of post-stroke neurological deficits is relatively difficult and biased in mice due to their small size, especially among non-clinicians or inexperienced researchers8,9. To address this, different groups have developed various scales to detect deficits. The most used and known scales include the 3-point Bederson scale (BS)10, the 5-point modified Bederson scale (mBS)11, the 5-point Longa score (LS) (which is similar to mBS)6, the modified Neurological Stroke Scale (mNSS)12,13, the 18-point Garcia scale (GS)9 and the more detailed DeSimoni14 or differently known as &#34;Neuroscore&#34; (NS)9,15 scale. Unfortunately, some of these scales are either too crude and are restricted only to the few acute-phase days following stroke (BS, mBS and LS)8,9 or their interpretation is blurred by general deficits (NS).
With this background, we previously developed and validated the mouse Stroke Unit (mSU) support protocol, along with the experimental stroke scale (ESS) for mice13. The rationale for mSU was to adapt knowledge from the clinical routine (i.e., human stroke units) into preclinical research of stroke, while ESS critically consolidated previously existing but &#34;insensitive&#34; or redundant stroke scales into a practical, refined, sensitive and time-efficient one. The mSU consists of frequent and adapted monitoring of mouse&#180;s basic clinical parameters with adapted application of animal support to enhance survival13.
Indeed, data from our laboratory and others verify the value of both methods. The mSU translates clinical basic supportive measures and advances7 from humans to mice, significantly reduces mortality of the fMCAo in mice from 60-70% to 10-15%13 thus allowing for studies of larger strokes, and can be effectively applied in independent laboratories since then16,17,18,19. In addition, the ESS can distinguish between focal (focal component of ESS, fESS) and general (general component of ESS, gESS) post-stroke deficits and symptoms, models the human clinical scoring (differentiation between focal and general signs and symptoms in humans), is linearly related to the stroke lesion size and is long-term sensitive to deficits13,20. Still, despite the proven value and efficacy of both mSU and ESS, the lack of clear-cut, visualized, standardized instructions for even inexperienced researchers leaves several open questions on mSU application and significant subjectivity on scoring focal deficits on fESS.
As such, our present article aims to provide video-assisted and clear instructions for mSU and fESS protocols. We strongly believe that it will support stroke researchers to increase the translational efficiency of their stroke studies, significantly reduce the loss of animals during the first 3-10 days after stroke, reduce experimentation costs, and eventually reproducibly evaluate neurological deficits for months after stroke13. Additionally, the add-on combination of the Ladder-rung test and Cylinder test can easily quantify the focal limb paresis (fore- and hindlimbs) due to fMCAo.
To showcase mSU and fESS efficiency, we provide medium- (14-days) and long-term (6-month) data on mice after fMCAo. Twelve-week-old male C57Bl/6J mice (n= 31) were used and housed under controlled temperature (22 &#177; 2 &#176;C), with a 12 h light-dark cycle period and access to pelleted food and water ad libitum. Mice were divided into two cohorts followed for 14 days (n=10, cohort 1) and 6 months (n=15, cohort 2) respectively. These mice were subjected to a 60 min cerebral ischemia using the well-described model of fMCAo13,20,21 under isoflurane anesthesia. Sham-operated animals (n=6, operated as the above two cohorts, but no ischemia was induced) followed for 6 months served as controls. Buprenorphine was used as a pre- and 3-day post-operative analgesic. The Ladder rung and cylinder tests were also performed for the 6-month cohort as a part of the neurological scoring.
All animals were checked daily for humane endpoints during the first 14 days post-stoke, defined as: 1) severe hypothermia (&#60;33 &#176;C) and/or immobility (e.g., score 4 on Test 6 of fESS or &#34;spontaneous activity&#34; test of gESS) that was not improved by &#34;mouse Stroke Unit&#34; treatment (i.e., passive heating and active feeding, see 1.4, 1.6, 1.8) within one hour, 2) signs of pain or anxiety behavior (e.g. score &#62;2 in &#34;anxiety/automatic behavior&#34; test of gESS), even after post-operative analgesia as per the protocol.
In this protocol, post-stroke support of the mice in the form of mSU begins immediately after mouse recovery from the fMCAo operation. This has 3 phases: phase A (0-48 h post-reperfusion), phase B (&#62;48 h and up to the &#34;end of needed active support&#34;, usually at day 10-14, depending on individual animal&#180;s phase B), and phase C (day 14 onwards). It has five significant interventions (visits/Risk Stratification Score, feeding, fluids, temperature, and local disinfections; see 1.1. to 1.8. below) tailored to each animal, according to each phase (A, B or C) and its daily actual clinical status assessed by the Risk Stratification Score (RSS), see Supplementary Figure 1 and Table 1 with three typical examples. The required materials and tools for mSU are shown in Figure 1a and described in Table of Materials. We also provide a template for animal monitoring during mSU (see Supplementary File 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tools for mSU</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5% Dextrose solution&#160;</td>
			<td>VIOSER S.A</td>
			<td>na (not applicable)</td>
			<td>Any genericon</td>
		</tr>
		<tr>
			<td>Contactless Digital Thermometer</td>
			<td>AVRON</td>
			<td>YTC20095</td>
			<td>Any genericon</td>
		</tr>
		<tr>
			<td>Digital weight scale</td>
			<td>KERN &#38; Sohn Gmbh</td>
			<td>FCB6K1</td>
			<td>Any genericon</td>
		</tr>
		<tr>
			<td>Food pellets</td>
			<td>Mucedola srl</td>
			<td>na</td>
			<td>Any genericon, use the normal food of your animal facility</td>
		</tr>
		<tr>
			<td>Heating Plate</td>
			<td>Photax</td>
			<td>na</td>
			<td>Photax dishwarmer 2</td>
		</tr>
		<tr>
			<td>Liquid antiseptic</td>
			<td>Sch&#252;lke &#38; Mayr GmbH</td>
			<td>na</td>
			<td>Octenisept&#174;</td>
		</tr>
		<tr>
			<td>Normal food blender</td>
			<td></td>
			<td>na</td>
			<td>For pellet pulverizing. Any genericon</td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>DEMO S.A.</td>
			<td>na</td>
			<td>Sodium Chloride Injection 0,9%</td>
		</tr>
		<tr>
			<td>Pinsetter and cotton buds</td>
			<td></td>
			<td>na</td>
			<td>Any genericon</td>
		</tr>
		<tr>
			<td>Sugar</td>
			<td></td>
			<td>na</td>
			<td>Any genericon</td>
		</tr>
		<tr>
			<td>Syringes (1ml) with 27-gauge needle</td>
			<td></td>
			<td>na</td>
			<td>Any genericon. For food administration (without needle) and for subcutaneous fluid administration (with needle)</td>
		</tr>
		<tr>
			<td>Tools for ESS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>45&#176; angled surface&#160;</td>
			<td>construct it</td>
			<td>na</td>
			<td>Made out of plexiglas or other material, with rubber-surface, for climbing tests</td>
		</tr>
		<tr>
			<td>cotton swab&#160;</td>
			<td>Any genericon</td>
			<td>na</td>
			<td>commercial ear cotton buds, make its cotton tip long and thinned</td>
		</tr>
		<tr>
			<td>edge-sharpened wooden stick</td>
			<td>Any genericon</td>
			<td>na</td>
			<td>e.g. toothpicks</td>
		</tr>
		<tr>
			<td>Long beam&#160;</td>
			<td>construct it</td>
			<td>na</td>
			<td>Dimensions: 1 x 1 x1cm, approximately 100cm long, wooden. Place between two table-edges for beam walking test</td>
		</tr>
		<tr>
			<td>Thick glove&#160;</td>
			<td>Any genericon</td>
			<td>na</td>
			<td>to prevent animal trauma when falling from beam</td>
		</tr>
	</tbody>
</table>

the mouse stroke unit protocol with standardized neurological scoring for translational mouse stroke studies

The filament model of middle artery occlusion (fMCAo) is perhaps the most translational mouse stroke model, allowing for controlled ischemia with intravascular reperfusion/recanalization. However, it lacks alignment with current clinical advances for stroke care (e.g., Stroke Units), usually employs subjective or vague neurological scoring among laboratories, and exhibits high acute-phase mortality. Here, we address these limitations with validated video-guided protocols. We present the mouse Stroke Unit (mSU) protocol with instructional videos and a decision algorithm (Risk Stratification Score), bridging the gap between clinical and mouse stroke modeling. To increase accuracy and sensitivity of stroke neurological scoring, we present for the first time a video-standardized format of the focal Experimental Stroke Scale (fESS) and prove its value up to 6 months post-stroke. Additionally, protocols for mice Ladder-rung test, as well as the known Cylinder test, for unbiased, quantitative assessment of limbs&#180; motor function are presented. Results highlight mSU&#39;s translational efficacy. Focal ESS (fESS) excels over other known scales in detecting focal stroke deficits, capturing recovery, and maintaining sensitivity for up to 6 months post-stroke. Ladder-rung and Cylinder tests objectively quantify and monitor fore- and hind-limb motor deficits, long-term. In summary, integrating mSU, fESS, and motor function tests provides a robust framework for clinically relevant stroke investigations. Our protocols improve the translational value in mouse stroke research.

The mSU, as described above, begins after the fMCAo operation. Our representative results in the two independent mouse cohorts of fMCAo stroke and sham (Figure 4a) confirms the previously proven value of mSU, particularly during the critical period between days 3 and 1013. In our cohorts, mortality occurred for 3/15 animals of Cohort 1 (stroked, 6 months follow-up), 2/10 of Cohort 2 (stroked, 14 days follow-up), and 0/6 of sham-operated animals (F...

This article presents a protocol for executing the highly translational and independently proven mouse Stroke Unit (mSU) concept in large-stroke mice. It also provides a standardized protocol for executing the focal experimental stroke scale in mice, which evaluates stroke focal deficits detected even long-term.

The Mouse Stroke Unit Protocol with Standardized Neurological Scoring for Translational Mouse Stroke Studies

The filament model of middle artery occlusion (fMCAo) is perhaps the most translational mouse stroke model, allowing for controlled ischemia with ...

the-mouse-stroke-unit-protocol-with-standardized-neurological-scoring

Research

JoVE Journal

Neuroscience

442 Views.  National and Kapodistrian University of Athens. This article presents a protocol for executing the highly translational and independently proven mouse Stroke Unit (mSU) concept in large-stroke mice. It also provides a standardized protocol for executing the focal experimental stroke scale in mice, which evaluates stroke focal deficits detected even long-term. 

Video: The Mouse Stroke Unit Protocol with Standardized Neurological Scoring for Translational Mouse Stroke Studies

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

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 ...

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume three-dimensionally. While MRI is an extremely sensitive tool for detecting tissue abnormalities, association of signal changes with an underlying pathological process is usually not straightforward. In the central nervous system, for example, inflammation, demyelination, axonal damage, gliosis, and neuronal death may all induce similar findings on MRI. As such, interpretation of MRI scans depends on the context, and radiological-histopathological correlation is therefore of the utmost importance. Unfortunately, traditional pathological sectioning of brain tissue is often imprecise and inconsistent, thus complicating the comparison between histology sections and MRI. This article presents novel methodology for accurately sectioning primate brain tissues and thus allowing precise matching between histology and MRI. The detailed protocol described in this article will assist investigators in applying this method, which relies on the creation of 3D printed brain slicers. Slightly modified, it can be easily implemented for brains of other species, including humans.

The overall goal of this protocol is to accurately align magnetic resonance imaging (MRI) image volumes with histology sections via the creation of customized 3D-printed brain holders and slicer boxes.

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...