Computational protocol: TRPV1-mediated Pharmacological Hypothermia Promotes Improved Functional Recovery Following Ischemic Stroke

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Protocol publication

[…] At 30 days post-stroke, brain samples from a parallel cohort of mice were collected and prepared for DTI scanning as described previously. Briefly, mice were transcardially perfused with 15 ml heparinized phosphate buffered saline (PBS, 20 unit/ml) followed by 15 ml 4% paraformaldehyde (PFA) for fixation. The mouse skull was exposed by removal of the skin, muscle, ears, nose tip, and lower jaw and fixed by 4% PFA at 4 °C overnight. After that, the head was incubated in 0.01% sodium azide (NaN3) in PBS at 4 °C for 7 days and then in 5 mM gadopentetate dimeglumine solution (Bayer HealthCare Pharmaceuticals Inc., Wayne, NJ) at 4 °C for 14 days. The head was equilibrated at room temperature for 6 hours before imaging.All DTI scans were acquired on a 9.4 T Bruker Avance Biospec Spectrometer, 21-cm bore horizontal scanner with a 35 mm volume resonator (Bruker BioSpin, Billerica, MA) with Paravision 5.1 software (Bruker Biospin, Billerica, MA). The 3D DTI scan parameters were as follows: Spin echo, b-value = 0 and 1000 s/mm2, 20 diffusion directions with one non-diffusion weighted image, TR = 500 ms, TE = 14.8 ms, FOV = 1.5 × 1.0 × 2.0 cm, matrix = 164 × 96 × 96, NEX = 1, δ = 3 ms, Δ = 7 ms.The image volumes were processed using DTI studio and ROIeditor (www.mristudio.org). The alignment was carried out using the DiffeoMap software,. The alignments were then applied to the tensor and the various maps (FA, colormap, B0, radial diffusivity), to yield aligned brain volumes. The inverse alignment transformation was applied to the template segmentation map, and then down-sampled to match the original subject resolution. This generated segmentation maps for the subject brains in their original scanned orientation and resolution. Fiber tracking was carried out using DTI Studio to quantitate fiber number and fiber length through specific brain regions (e.g. the thalamus). Amira (version 5.6; FEI) was utilized for presentation-quality visualization of FA maps and fiber tracking data. [...] Histology necrosis data was expressed as mean ± SEM. A t test was used for comparison between Stroke/vehicle and Stroke/DHC groups at each position. Pearson correlation analysis was used to evaluate the correlation between thalamic injury and cortical necrosis injury at each position from bregma in Stroke/vehicle group. Parallel line analysis was used to determine if the correlation of thalamic injury and cortical injury were significantly different in Stroke/vehicle and Stroke/DHC groups. Cell counting data was expressed as median ± SEM. A Kruskal–Wallis one-way ANOVA test was used to evaluate differences among three groups and a Mann–Whitney U test with Bonferroni correction was used for post hoc multiple comparisons. A t-test was used to compare fiber tract number between groups. All statistical calculations were performed using SigmaPlot 12.5 (Systat software). Behavioral data was expressed as mean ± standard error of the mean (SEM). Two-way repeated measure ANOVA was used for comparing groups with repeated measures. The Holm-Sidak method was used for all pairwise multiple comparisons. […]

Pipeline specifications

Software tools ParaVision, MRI Studio, SigmaPlot
Applications Miscellaneous, Magnetic resonance imaging
Organisms Mus musculus
Diseases Cerebral Infarction, Wounds and Injuries, Stroke