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

Summary

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.

Abstract

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´ motor function are presented. Results highlight mSU'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.

Introduction

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.<....

Protocol

The experiments reported in this article were conducted following National and European guidelines for the use of experimental animals and were approved by the Greek governmental committees (Athens, License No "843895_06-09-2022").

1. The "mouse Stroke-Unit" (mSU)

1. Assess animal´s clinical status daily ("visits") and calculate its RSS score
   1. Print the Supplementary File 1, preferably before animal´s surgery, for recording data and monitoring.
   2. Assess each animal clinically at standard time points (visits): ea....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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