Mice expressing soluble decoy VEGF receptor 3 (sVEGFR3) in an LDLR/ApoB background, which reduces the formation of lymphatic vessels in the heart and other parts of the body, formed a transgenic group (TG). Wild-type (WT) littermates were selected for the control group.6. Permanent MI was induced in 16 TG and 23 WT mice by ligating the left anterior descending (LAD) coronary artery.30,31. Briefly, rats were anesthetized with isoflurane (Univentor-400, Univentor, Zejtun, Malta) 4% inhalation before LAD operation and anesthesia was maintained with 2% during the operation. The heart was exposed and pushed out of the thorax and the LAD was sutured approximately at the medial level of the heart using a 6-0 silk suture (Mansfield, MA, USA). After LAD ligation, the heart was returned to its original position. After surgery, rats were treated with buprenorphine (0.3 mg/ml) (Temgesic (RB Pharmaceuticals, Slough, UK)) 0.05–0.1 mg/kg and carprofen (50 mg/ml) (Rimadyl (Pfizer Oy Animal Health, Helsinki, Finland)). found.)) 5 mg/kg subcutaneously for analgesia. Analgesia was repeated on the first and second day after surgery. All animal experiments were performed in accordance with ARRIVE guidelines as well as guidelines for the use and care of laboratory animals approved by the University’s Institutional Animal Care and the Finnish National Animal Experiment Board. Protocols were carried out and animal experiments were carried out in accordance with the Act on the Protection of Animals. Number of animals used for scientific or educational purposes (497/2013).
Images of mice before MI operation (day 0) and 3 days (n = 11 in TG group, n = 14 in WT group), 7 days (n = 8 in TG group, n = 9 in WT group), 21 days was destroyed. (n = 8 in TG group, n = 11 in WT group) and 42 days after MI (n = 1 in TG group, n = 4 in WT group). The number of animals due to death varied between time points. MRI experiments were performed using a horizontal 9.4 T magnet (Varian Inc. Palo Alto, CA, USA) controlled by a Bruker Biospec console (Bruker GmbH, Ettlingen, Germany). A quadrature volume transceiver coil with an inner diameter of 35 mm was used (Rapid Biomed GmbH, Ettlingen, Germany). For MRI experiments, rats were anesthetized with 4% isoflurane mixed with oxygen and nitrogen gases in a 1:3 ratio. Isoflurane levels were reduced to 1–1.5% during imaging. The body temperature of the mouse was maintained close to the natural temperature level (37 C°) by a warm water pad placed under the mouse. ECG was measured using needle electrodes from the forepaws and respiration was controlled with a pneumatic pillow placed under the rat’s abdomen. Both signals were registered using a Model 1025 (Small Animal Instruments Inc., NY, USA) and were used to gate all MRI experiments.
Multislice short-axis ECG-triggered and respiration-gated waveform images covering the whole heart were acquired using gradient-echo-based fast imaging with a steady-state pressure (FISP) readout sequence. Imaging parameters for Sunny images were field of view (FOV) = 4 × 4 cm.2, slice thickness = 1 mm, matrix size = 192 × 192, echo time (TE) = 1.9 ms, repetition time (TR) = 8.0 ms, scan TR = 99.0 ms, flip angle = 10° of number of frames. There were 10-11 depending on the heart rate of the mouse. Depending on the size of the heart, 8–12 slices were imaged.
RAFFn relaxation times (TRAFFn) was obtained by applying the two preparation modules RAFF2 or RAFF4.26,27 The waveforms of the pulses (called RF power (γB1/(2π)) of 1250 Hz and 648 Hz, respectively, and a period of 1.13 ms.27). Pulse trains with durations of 0, 9.1, 18.1 and 36.2 ms were added resulting in four separately weighted images for both RAFF2 and RAFF4. Regardless of the duration of the pulse train, the acquisition occurred in the same cardiac phase.
T1 p Data were acquired using a rotating frame preparation module consisting of an adiabatic half-passage (AHP) pulse (power = 1250 Hz, duration = 3.0 ms), continuous-wave spin-locked pulse (power = 625 Hz). Four different weighted images were obtained with spin-lock times (TSL) of 0.4, 9.4, 27.4 and 45.4 ms. After the spinlock pulse, an AHP back pulse (power 1250 Hz, duration 2.0 ms) was applied.23. A single cardiac phase readout occurred for all weighted images.
T2 Measurements involved a Hahn double-echo preparation consisting of an AHP excitation pulse (power = 1250 Hz, duration = 3.0 ms), two hyperbolic secant 1 (HS1)-pulses.32 (power = 1250 Hz, duration = 4.5 ms) and an AHP back pulse (power = 1250 Hz, duration = 3.0 ms). Coherent delays were applied between pulses, resulting in total TEs of 0.05, 2.3, 4.5, and 14.0 ms, resulting in four different weighted images. Delay in front of t2 Preparations were also included as in RAFFn and T1 p.
B1 Measured by applying a hard pulse with 625 Hz power and pulse durations of 0, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5 and 1.75 ms.33.
The FISP-readout sequence was used to obtain a single short-axis slice with the same geometry at the mid-ventricular level for all resting times and B .1 Measure with the following parameters: FOV = 4 × 4 cm2Slice thickness = 1 mm, matrix size = 256 × 256 (for b1 The size of the measurement matrix was 128 × 128, TE = 1.9 ms, TR = 14.9 ms, the scan was dependent on both heart and respiratory rates. The minimum delay between weight pulses was chosen as 1460 ms to speed up the imaging time while signal loss from steady state was still reasonable.
Hearts were perfused through the LV with phosphate-buffered saline and then immersed in sucrose containing 4% paraformaldehyde. After 4–16 h, the liquid was changed to 15% sucrose. The hearts were then paraffin embedded and 4 μm thick cross sections of the heart were stained with hematoxylin eosin (HE) and Sirius red (SR) to differentiate the fibrotic and collagen areas of the infected myocardium.8. Histological sections were photographed with a light microscope (Nikon Eclipse, Ni-E, Tokyo, Japan). Histology was performed on day 21 and day 42 after LAD ligation.
All MRI images were analyzed using Aedes software (http://aedes.uef.fi/) in Matlab (Mathworks Inc. Natick, MA, USA). Rest time (TRAFF2TRAFF4T1 p And T2) and B1 Maps were reconstructed from signal intensities in a pixel-by-pixel manner using Addis software. All maps were fitted to logarithms of signal intensities using a linear function. Regions of interest (ROI), which included the MI and distal regions, were manually traced with visual delineation of the MI and distal regions. An overlay ROI was used to ensure that the enhanced relaxation times were specific to the region of the myocardium. The overlay ROI was created based on the raw weighted MR image. Additionally, audio and histology images were used to localize the fibrotic MI area on resting-time maps. End-systolic volume, end-diastolic volume, ejection fraction (EF), and stroke volume were defined based on the apparent endocardial border in cine images.23.
Infarct percentage was analyzed by a method based on manually traced midline length with a function (L(infect)/L(Circumference)) × 100%, where L(infect) Indicates the measured arc length of any T to MI area.RAFF2TRAFF4T1 pT2SR or HE stained section and L(Circumference) represents the midline length of the entire myocardium.34. Area of difference (AOD) with respect to T2– Defined MI area (A2), which includes both infarct and edema areas, is calculated ((A2 – A)/A) × 100%, where A denotes the area of infarct size without edema at any T;RAFF2TRAFF4or T1 p Rest Time Map35. Furthermore, the area of LV edema outside the exact infarct region in SR-stained sections was calculated with the open-source ImageJ (National Institutes of Health, MA, USA) software as the extracellular space between myocytes within the entire left myocardium. Calculated. After this calculation, the AOD was calculated similarly to the MRI data. Co-registration between MRI and histological sections was based on visual agreement.
All numerical values are given as mean ± standard error of the mean (SEM). A two-way ANOVA with a Bonferroni post hoc test for multiple comparisons was applied to compare spatial and temporal changes between infarcted and distal regions of the myocardium. Pearson’s correlation was calculated to examine the relationship between MRI relaxation time maps and histology sections. Analysis was performed using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA).
Ethics approval and consent to participate.
All surgical procedures were performed in accordance with protocols approved by the Finnish Committee for the Use and Care of Laboratory Animals.