Ultrafast Doppler imaging and ultrasound localization microscopy reveal the complexity of vascular rearrangement in chronic spinal lesion | Scientific Reports – Nature.com

41598 2022 10250 Fig1 HTML ProxyEgg Ultrafast Doppler imaging and ultrasound localization microscopy reveal the complexity of vascular rearrangement in chronic spinal lesion | Scientific Reports - Nature.com

All experiments performed in this study were in accordance to the French and European Community Council Directive of September 22 (2010/63/UE). They were also approved by the local Institutional Animal Care and Ethics Committees (#59, ‘Paris Centre et Sud’ project #2018-05 and Sorbonne University project #1514.01). Accordingly, the number of animals in our study was kept to the minimum necessary. We established in a preliminary experiment that N = 5 was the smallest number of animals required per group to detect statistical difference in our imaging experiments. Finally, all methods are in accordance with ARRIVE guidelines.

Surgical preparation and SCI model

Animals arrived in the animal facilities at the IBPS institute 2 weeks before the beginning of experiments. Fifty adult female Wistar rats (225–250 g) were obtained from Janvier labs (France) and housed under controlled temperature (22 ± 1 °C), relative humidity (55 ± 10%) and 12 h light–dark cycle. Before and after surgical interventions food and water were available ad libitum.

Surgical procedures were performed under reversible, continuous Isoflurane anesthesia (2–2.5%, Isofluran®) and sterile precautions were used throughout. In order to perform the spinal cord contusion, skin and musculature was cut from T7–T10 and the dorsal surface of T8–T9 exposed by laminectomy, with the dura remaining intact. The vertebral column was stabilized by fixing vertebral bodies rostrally and caudally to the impact area with clamps attached to the base of the Benchmark impactor device. A 2.5 mm diameter tip was delivered from 7 mm height with a speed of 1.96 m/s over the spinal cord dorsal surface, and contact time after the impact was 1 ms. These parameters lead to a severe lesion with pronounced locomotor deficits evaluated using the Basso, Beattie and Bresnahan locomotor rating scale (initial BBB score 1 day after the trauma between 0–0.5).

To prevent urinary infections, rats received subcutaneous injections of eurofloxacin (Baytril® 10%) once a day during the first week post-lesion. Until restoration of normal micturition, bladders were manually emptied twice a day, and the health state of operated rats monitored by regular weight and visual inspection of the surgical wound.

The rats were randomly assigned into three groups, five animals per group: intact (received a sham lesion, where the muscles were opened but the spinal cord left intact), 4- and 8-weeks post-injury. BBB locomotor scores were taken at days 1, 7, and then weekly for a total of 7 weeks. Since the animals were transported to another institute for spinal cord imaging, recording of BBB scores was stopped 1 week before the experiment. At the end, the rats were sacrificed for histological investigation.

Surgical procedures and preparation for imaging

Under deep anesthesia (IP) bolus of Medetomidine (Domitor, 0.4 mg kg−1) and ketamine (Imalgène, 40 mg kg−1), a laminectomy (centered on the spinal cord lesion) was performed between thoracic T6 and lumbar L2 vertebrae (two vertebrae above and below the initial laminectomy), thus opening a window allowing positioning of the entire ultrasonic probe (14 mm) in a sagittal plane.

Thereafter, the animal was placed on a spinal cord stereotaxic frame. Anesthesia was maintained but reduced, using subcutaneous perfusion of Medetomidine (0.2 mg/kg/h) and ketamine (25 mg/kg/h) using a syringe pump. During the surgical procedure and the imaging session, the animal’s body temperature was kept at 37 °C using a heating blanket and an intrarectal probe (Physitemp, USA), and heart and respiratory frequencies were monitored (MouseOxPlus, Ugo Basile, Italy). Each imaging session lasted 2–3 h.

Ultrafast Doppler imaging and signal analysis

Two milliliters of saline were gently dropped on the spinal cord (the dura mater was kept intact), and the window created by laminectomy was then filled with echographic gel. The ultrasonic probe (f = 15 MHz, 100 µm spatial pitch, 128 elements, Vermon, France) was positioned just above the window using a 3-axis motor. The probe connected to an ultrasonic ultrafast imager (Iconeus, 128 channels, 62.5 MHz sampling rate) was driven with a prototype software (Iconeus, Paris, France, and Inserm Accelerator of Technological Research in Biomedical Ultrasound, Paris, France). The imaging session started by a 3D scan of the spinal cord that allowed positioning of the probe. By symmetry of the vascular structure, the median plane was deduced and the alignment of the probe with the spinal cord adjusted by rotating it until the anterior spinal artery (ASA) was entirely seen on the images. Ultrafast Doppler at 5500 Hz PRF for relative CBV, Ultrafast Doppler at 20,000 Hz PRF for blood flow direction and at 5000 Hz PRF for ultrasound localization microscopy acquisitions were performed successively.

Measure of SBV using Power Doppler imaging

SBV values were obtained with the ultrafast Doppler imaging method, which consists of compounded plane-wave ultrasound transmissions48. Thus, each frame was a compound plane wave frame resulting from the coherent summation of 11 compounded tilted plane waves with angles separated by 2° and ranging from − 10° to 10°. The framerate used was 500 Hz, resulting in a 5500 Hz pulse repetition frequency (PRF). The Singular Value Decomposition (SVD) clutter filtering (Demene et al. 2015) typically used for brain tissue had to be modified for our use on spinal cord tissue. The SVD thresholds were automatically determined thanks to adaptive spatiotemporal SVD using the similarity of spatial singular vectors49. Over 400 compounded frames, the first 30 singular values were excluded to remove the tissue and the last 230 were excluded to remove the noise. The resulting 400 successive images were averaged to obtain a single SBV image that was analyzed by spatial averaging of the different ROI drawn using MATLAB 2020a.

Choice and size of areas taken for the measurements of SBV, directions of flow, density of blood vessels and bubble velocity

For the quantification (of the SBV, density of blood vessels and bubble velocity) in the lesion, rostral and caudal to the lesion: three subparts of equivalent area (30 mm along the antero-posterior axis X 18–20 mm dorsal to caudal, i.e. 530 mm2) of the thoracic spinal cord were drawn: at the lesion site (around the lesion epicenter), rostral or caudal to it. In intact animals, the same areas were taken, the area at the center was taken for the counter part of the lesion side, and, as in lesioned animals, the same 530 mm2 rostral and caudal to the center were taken for the rostral and caudal values. The respective values were computed on MATLAB.

Quantifications of bubble velocity in the ‘whole cord’ (Fig. 5), an equivalent rectangle surrounding the entire thoracic cord imaged (or most of it) of the following dimensions 90 mm (Rostro-caudal) X 18–20 mm (dorso-ventral axis) was computed on MATLAB in all animals included in our study.

The same principle was used for all the other measures: an equivalent area was defined in all groups, surrounding either the dorsal/ventral thoracic cord (Fig. 3), CSA, ASA, dorsal vein (Figs. 4, 5).

Direction of flow

A different acquisition sequence was used to analyze the direction of flow. Each frame was a compound image of 5 angles (− 4°, − 2°, 0°, 2° and 4°), which together with a sampling frequency increased to 4000 Hz resulted in a 20,000 Hz PRF. The total acquisition contained 2000 frames and lasted 0.5 s. The same SVD filter as for the Power Doppler acquisition was used to separate the tissue signal from the blood signal. Using the same spectral analysis as in the color Doppler images50, the SBV signal was separated into two series of images representing the SBV going toward and away from the probe. After averaging the 2000 frames of the two directional SBV, the general directional SBV was obtained by subtracting the SBV going upward from the SBV going downward, creating a directional power image. The images were then spatially averaged in the same ROIs drawn for the analysis of the SBV previously described.

Ultrasound localization microscopy (ULM)

A catheter filled with saline was inserted in the rat jugular vein before positioning the animal on the stereotaxic frame. After all previously mentioned measures, ULM was performed similarly to the methods described in42 using the same ultrasound imager as above. Compounded frames were acquired at a 1000 Hz framerate (with angles at − 4°, − 2°, 0°, + 2°, + 4°, PRF = 5000 Hz) using the same system (Iconeus One, Iconeus, Paris, France) as above for a total time of 150 s. Beamformed data were filtered using the SVD spatio-temporal filter described in51, and the 7 first singular values were removed to extract microbubble signals from the surrounding tissues. Microbubbles were detected as the brightest local maxima in the images. Tracking of the maxima positions was performed using the Iconeus software (Iconeus, Paris, France, and Inserm Accelerator of Technological Research in Biomedical Ultrasound, Paris, France) and gives all positions in space (x-axis and z-axis positions) and time (frame number) for each detected individual bubble.

The successive positions gathered in one track were used to compute the interframe bubble velocity vector components (along probe x-axis and depth z-axis), and absolute velocity magnitude.

Density maps were computed by counting all the microbubbles that passed through each pixel during acquisitions. Since the total number of bubbles injected is not exactly the same from one injection to another, comparison of raw density between animals would reflect this random effect instead of intrinsic differences between groups. To avoid this variability, the value of density was normalized by the total number of bubbles detected in the ROI contouring the whole spinal cord. To compare these maps between animals and consider the variability coming from the random number of bubbles injected into the blood stream, the value of density was normalized by the total number of bubbles detected in the spinal cord imaged.

Analysis of the density of blood vessels using the ULM analysis was performed using Image J. The images obtained at the previous steps were first transformed as black and white pictures. Using a common threshold (131) for all animals, the percentage of staining occupied by the blood vessels was measured in each animal in the six following compartments: Lesion site, rostral or caudal to the lesion site, the ASA or the CSA at the level of the lesion site. For each compartment, the area measured for all animals was similar. Results are expressed as percentage of area occupied by the staining.

The vessel tortuosity index was computed thanks to the MATLAB function “vessel_tortuosity_index” written by Maz M. Khansari52. For each animal, three vessels located rostral, caudal and the last one close to the lesion, were chosen on the density map given by the ULM algorithm. A line was drawn over each one to use it as an input for the function. These values were averaged to obtain the tortuosity index for each animal.

Each pixel of the velocity maps was computed as the mean velocity of every micro-bubble detected in this pixel during the whole acquisition. These two maps were then spatially averaged in ROIs drawn the same way as for the SBV analysis.

Statistical analysis

For each type of statistical comparison, the same protocol of hypothesis verification was applied to choose the appropriate statistical test. First, the two assumptions needed for parametric tests were tested. The normality of the data distribution of each group was tested with a Lilliefors test and the equality of the variances was tested with a Bartlett’s test.

Then, an ANOVA was performed to determine if a difference between two groups could be detected with a statistical test. If one of the two conditions for the parametric tests was not met, the Kruskal–Wallis test was performed for the same purpose. If the ANOVA detected a statistically significant difference, pair wise tests were used to determined which groups differ. For data suited for parametric test, a student test was used. A Wilcoxon rank sum test was used otherwise.

In Fig. 7, for the double correlation matrix: Considering that some measures did not have a gaussian distribution (and were therefore not compatible with a parametric test), all correlations were computed using Spearman coefficients, (which is the non-parametric counterpart of the Pearson’s Linear Correlation Coefficient). The p values corresponds to the probability of rejection of the null hypothesis that the correlation is equal to zero. All panels of Fig. 7 were plotted using GraphPad.

Statistical analysis was performed using Matlab. Graphs were made using GraphPad.

Perfusion and immunohistochemistry

At the end of the experiment, rats were administered a lethal dose of pentobarbital (Euthasol® 400 mg/mL; 150 mg/Kg), and were transcardially perfused with saline 9‰ at 37 °C, then with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. PFA-fixed tissues were incubated in 15% and 30% sucrose for 24 h, and 48 h at 4 °C, respectively, then embedded in optimal cutting temperature compound (OCT; Tissue-Tek), quickly frozen in a dry ice/isopentane bath and stored at − 80 °C. 40 μm sagittal sections were cut on a cryostat (Leica CM3050 S) and mounted on glass slides (Superfrost® Plus).

For immunolabeling, sections were permeabilized with 0.3% Triton X_-100 in PBS during 5 min at room temperature, and incubated for 1 h in 10% bovine serum albumin (BSA)/PBS. Blocking was followed by incubation overnight at room temperature with the primary antibodies diluted in 5% BSA/PBS. The following primary antibodies were used: rabbit glial fibrillary acidic protein (GFAP; Dako, 1:2000), mouse-SMI 71 (Covance, 1:100), rabbit-a-laminin (Sigma, 1:100). Finally, appropriate secondary antibodies (Alexa Fluor 488 and 555 conjugated, 1:1000; Life technologies) diluted in BSA 5% were used.

Figures showing large longitudinal spinal cord sections were produced using a Zeiss Axio Zoom V16 microscope with image stitching and were processed using ImageJ (NIH, USA).

Quantification of SMI-71 positive cells

Pictures of 4 sections/rat (160 µm between each section) stained with anti-SMI 71 antibody were taken for quantification. With ImageJ, a rectangle of 1.6 mm2 was drawn at the level of lesion, as well as at the level of the caudal and rostral part of each section. After fixing the same threshold for each section, particle analysis was performed and the mean value per rat was calculated and compared as total area between each group (intact-uninjured, 4 and 8 weeks post-injury; 3 rats per group). One-way Anova was done to compare values for each region between groups.

Source of this news: https://www.nature.com/articles/s41598-022-10250-8

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