Dataset on stroke infarct volume in rodents: A comparison of MRI and histological methods

This dataset offers images of mouse brains impacted by photothrombotic stroke in the sensorimotor cortex published by Weber et al. NeuroImage (2024). Data is gathered using two primary techniques: (1) whole-brain ex-vivo magnetic resonance imaging (MRI) and (2) 40 µm thick coronal histological sections that undergo immunofluorescence staining with NeuroTrace. Infarct areas and volumes are assessed through MRI at two distinct time frames—three days (acute) and 28 days (chronic) following photothrombotic stroke induction. Subsequently, the brains are sectioned into 40 µm thick coronal slices, stained with NeuroTrace, and imaged as whole sections. The dataset holds considerable value for reuse, particularly for researchers focused on stroke volume estimation methods as well as those interested in comparing the efficacy of MRI and histological techniques.


a b s t r a c t
This dataset offers images of mouse brains impacted by photothrombotic stroke in the sensorimotor cortex published by Weber et al. NeuroImage (2024).Data is gathered using two primary techniques: (1) whole-brain ex-vivo magnetic resonance imaging (MRI) and (2) 40 μm thick coronal histological sections that undergo immunofluorescence staining with NeuroTrace.Infarct areas and volumes are assessed through MRI at two distinct time frames-three days (acute) and 28 days (chronic) following photothrombotic stroke induction.Subsequently, the brains are sectioned into 40 μm thick coronal slices, stained with NeuroTrace, and imaged as whole sections.The dataset holds considerable value for reuse,

Value of the Data
• The dataset offers 2D NeuroTrace-stained brain images and full brain ex-vivo MRI images from mouse stroke tissue at acute (3 days post injury) and chronic (28 days post injury) time points.• This dataset contains manual lesion segmentation and automated volume estimation of ischemic brain sections from a total of 10 animals, done and validated by three experts.• We observe lesion volumes that are highly correlated, whether using full brain MRI images or sliced NeuroTrace-stained brain sections at acute and chronic time points.• This data can be employed by neuroscience researchers to detect and quantify ischemic lesions.Furthermore, the data enhance studies on stroke treatment and brain imaging techniques.• With high reuse potential, the data could validate new imaging quantification methods and inform computational models.

Background
Stroke volume plays a crucial role in determining the severity of infarcts and is an important measure for assessing treatment efficacy in preclinical animal models [2][3][4][5] .However, accurate estimates of stroke infarct volume can be challenging and can considerably vary between studies [6] .Several methods are suitable to investigate the extent of brain damage in experimental stroke models including magnetic presonance imaging (MRI) and histological staining.We therefore generated datasets of (1) whole brain ex-vivo magnetic resonance imaging (MRI) and ( 2) brain sections processed with immunofluorescence staining from stroked mice at acute (3 days) and chronic (28 days) time points after photothrombotic stroke to establish a semi-automated toolkit for more accurate and streamlined stroke volume estimation [1] .

Experimental Design, Materials and Methods
Methods for measuring lesion volume were compared utilizing histology and MRI from the brains of mice ( n = 10) that had undergone cortical ischemia.All procedures adhered to governmental, institutional (University of Zurich), and ARRIVE guidelines and were approved by the Veterinarian Office of the Canton of Zurich (ethics approval code: 209/2019).A total of 10 adult (12-15 weeks) female C57BL/6 mice were utilized ( n = 6 for acute timepoint, n = 4 for chronic timepoint).Previous studies showed similar stroke pathology between male and female mice after photothrombotic stroke [7] .C57BL/6 mice were bred at the Laboratory Animal Services Center (LASC) in Schlieren, CH.The mice were housed in standard type II/III cages with a 12 h day/light cycle (lights on at 6:00 A.M.), and they had ad libitum access to food and water.Prior to the experiment, all mice underwent a minimum one-week acclimatization period.All animals received a large photothrombotic stroke to the right sensorimotor cortex.At 3 and 28 days after injury induction, whole heads were collected, formalin-fixed and imaged using T2-weighted MRI.
Brains were removed, dissected, and histologically analyzed.We chose the timepoints based on previous literature [8][9][10] ; animals were categorized according to the phase of stroke as acute ( < 3 days post-stroke) or chronic ( ≥28 days post-stroke).

Photothrombotic Stroke Induction
Anesthesia was performed using isoflurane (5% induction, 1.5-2% maintenance, Attane, Provet AG).Novalgin (1mg/ml) was applied via drinking water; 24 h prior to the procedure and for three consecutive days directly after stroke surgery.Cerebral ischemia was induced by photothrombotic stroke surgery as previously described [ 2 , 9-13 ].In brief, the animals were secured in a stereotactic frame (David Kopf Instruments) and the surgical site was sanitized using betadine (Mundipharma, Germany).Next, the skull was exposed through a midline incision and a cold light source (Olympus KL 1,500LCS, 150 W, 3020 K) was positioned over the right forebrain cortex (anterior/posterior: -1.5 -+ 1.5 mm, medial/lateral: 0 mm to + 2 mm relative to bregma).Rose Bengal (15 mg/ml, in 0.9 % NaCl, Sigma) was intraperitoneally injected 5 min before illumination, and the region of interest was illuminated through the intact skull for 10 min.The incision was closed with and the animals were allowed to recover.

Sample Preparation and MRI Protocol
Animals were euthanized using pentobarbital (i.p, 150 mg/kg body weight, Streuli Pharma AG) and transcardially perfused with Ringer solution (containing 5 ml/l Heparin, B. Braun) followed by paraformaldehyde (PFA, 4%, in 0.2 M phosphate buffer, pH 7).For MRI procedure, whole mouse heads were collected and post-fixed in 4% paraformaldehyde (PFA) solution for 36 h.T2-weighted images were acquired on a 7T small animal scanner with 16 cm bore size (Bruker, Ettlingen, Germany).The fixed brains were put into an Eppendorf cap filled with perfluoropolyether (Fomblin Y, Sigma-Aldrich, Switzerland) and imaged using a cryogenically cooled quadrature surface coil (Bruker, Fällanden, Switzerland).A package of 20 slices with 0.3 mm thickness (no interslice gap) was acquired with a FLASH sequence with a field of view of 15 mm x 15 mm and matrix size of 300 ×240, yielding a spatial in-plane resolution of 50 μm x 50 μm (echo time TE = 10 ms, repetition time TR = 400 ms, 10 repetitions, total scan time 10 min 40 s).

Data Processing
Each fluorescence brain slice was registered to the corresponding ex vivo MRI slice using a 2D affine transformation.We used a tool provided by Fiji (Landmark Correspondences) [15] .Landmarks for registration were selected manually by a qualified researcher.Lesion area was manually delineated on each coronal brain section (MRI images and NeuroTrace-stained images) using FIJI ( ImageJ , version 2.1.0/1.53c).Numerical values for the number of pixels in the selection were obtained, converted into mm 2 and imported into MATLAB (R2022a, The Mathworks, Natick, MA, USA).Lesion volumes were calculated using a customized script.The modified akima interpolation (makima) function was used to interpolate the missing values.The area under the curve (AUC) was calculated using the trapezoidal computation rule.The volume was calculated by multiplication of the lesion area and the distance between the sections as follows: represents the cross-sectional area at x .For step-by-step guidance, please refer to our protocol ("Proto-col_data_postProcessing.docx").Neurotrace-stained sections were visualized using an Axio Scan Z.1 slide scanner (Carl Zeiss, Germany) with a 20x/0.8objective lens.Each histologically processed slice could be matched with the corresponding ex vivo MRI slice using the mouse brain atlas.

Statistics
Statistical analysis was performed using R-Studio.Sample sizes were designed with adequate power according to previous studies.All data were tested for normal distribution by using the Shapiro-Wilk test.The significance of mean differences between normally distributed data (MRI vs. Histology) were tested for differences with a two-tailed paired two-sample t-test.The significance of mean differences between two different timepoints (acute vs. chronic) was tested for differences using an unpaired two-sample t-test or a one-way ANOVA with post-hoc analysis (p adjustment method = holm), in case of multiple comparisons.Variables exhibiting a skewed distribution were transformed, using natural logarithms before the tests to satisfy the prerequisite assumptions of normality.To assess the relationships between MRI-based and histology-based assessments, correlation analysis was performed using the Pearson correlation coefficient (r).Data are expressed as means ± SD, and statistical significance was defined as * p < 0.05, * * p < 0.01, and * * * p < 0.001.

Limitations
This study provides snapshots of stroke volumes at two distinct timepoints, 3-and 28-days post-stroke injury induction.The absence of intermediate data points means we cannot delineate the trajectory of stroke volume changes over time.

Ethics Statement
All procedures were conducted in accordance with governmental, institutional (University of Zurich), and ARRIVE guidelines and had been approved by the Veterinarian Office of the Canton of Zurich (ethics approval code: 209/2019).In total, 10 adult female C57BL/6 mice were used.Breeding of C57BL/6 mice was performed at Laboratory Animal Services Center (LASC) in Schlieren, CH.All animals were housed in standard type II/III cages on a 12 h day/light cycle (6:00 A.M. lights on) with food and water ad libitum.All mice were acclimatized for at least a week to environmental conditions before set into experiment.