Name: The Mouse Stroke Unit Protocol with Standardized Neurological Scoring for Translational Mouse Stroke Studies (Video) | JoVE Journal
Uploaded: 2025-02-07T12:20:00.000+00:00
Duration: 10 min 45 s
Description: Learn the mouse Stroke Unit protocol for standardized neurological scoring in translational stroke research using intravascular occlusion models. This method enables reliable long-term assessment of focal deficits and recovery.

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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/pubmed?term=((Stroke%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Stroke.</a> Lancet. 371 (9624), 1612-1623 (2008).</li>
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<li>Bendele, A. M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBendele%2C+AM+%22Urinary+system%22">Urinary system</a>. Jones, T. C., Hard, G. C., Mohr, U. , Springer Berlin Heidelberg. Berlin, Heidelberg. 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/pubmed?term=((Cns+plasticity+and+assessment+of+forelimb+sensorimotor+outcome+in+unilateral+rat+models+of+stroke%2C+cortical+ablation%2C+parkinsonism+and+spinal+cord+injury%5BTitle%5D)+AND+%22Neuropharmacology%22%5BJournal%5D)">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/pubmed?term=((Deeplabcut%3A+Markerless+pose+estimation+of+user-defined+body+parts+with+deep+learning%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Relationship+of+national+institutes+of+health+stroke+scale+to+30-day+mortality+in+medicare+beneficiaries+with+acute+ischemic+stroke%5BTitle%5D)+AND+%22J+Am+Heart+Assoc%22%5BJournal%5D)">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>
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<li>Groger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Release+of+bradykinin+and+expression+of+kinin+b2+receptors+in+the+brain%3A+Role+for+cell+death+and+brain+edema+formation+after+focal+cerebral+ischemia+in+mice%5BTitle%5D)+AND+%22J+Cereb+Blood+Flow+Metab%22%5BJournal%5D)">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/pubmed?term=((Behind+the+wall-compartment-specific+neovascularisation+during+post-stroke+recovery+in+mice%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">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/pubmed?term=((Microanatomical+study+of+pyramidal+neurons+in+the+contralesional+somatosensory+cortex+after+experimental+ischemic+stroke%5BTitle%5D)+AND+%22Cereb+Cortex%22%5BJournal%5D)">Microanatomical study of pyramidal neurons in the contralesional somatosensory cortex after experimental ischemic stroke.</a> Cereb Cortex. 33 (4), 1074-1089 (2023).</li>
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</ol>

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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><tbody><tr><td>&lt;strong&gt;Tools for mSU&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>5% Dextrose solution&amp;nbsp;</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 &amp;amp; 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&amp;uuml;lke &amp;amp; Mayr GmbH</td><td>na</td><td>Octenisept&amp;reg;</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>&lt;strong&gt;Tools for ESS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>45&amp;deg; angled surface&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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 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 filament model of middle artery occlusion (fMCAo) is perhaps the most translational mouse stroke model, allowing for controlled ischemia with ...

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Research

JoVE Journal

Neuroscience

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

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Learn the mouse Stroke Unit protocol for standardized neurological scoring in translational stroke research using intravascular occlusion models. This method enables reliable long-term assessment of focal deficits and recovery.

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

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