Ultrasound and photoacoustic multimodal imaging are used in rodent brains after craniotomy

Ultrasound and photoacoustic multimodal imaging are used in rodent brains after craniotomy

Original title：A Skull-Removed Chronic CranialWindow for Ultrasound and
Photoacoustic Imaging of the
Rodent
Brain

Original author：Xuanhao Wang, Yan Luo, Yuwen Chen, Chaoyi Chen, Lu Yin, Tengfei Yu, Wen Heand Cheng Ma

"Ultrasound and photoacoustic imaging are becoming powerful tools for studying brain structure and function. Cranial passes introduce significant ultrasound signal distortion and attenuation leading to deterioration of image quality. For biological studies using rodents, image quality is often improved by craniotomy. However, craniotomy requires maintaining a long-term cranial window to prevent repeat surgeries. The authors propose a mouse model to eliminate acoustic obstruction at the top of the skull while producing minimal physiological perturbation to the imaging object. Using the new mouse model, no craniotomy was required before each imaging experiment. Three imaging systems confirmed the validity of our method: photoacoustic computed tomography, ultrasonography, and photoacoustic mesoscopy. Functional photoacoustic imaging of mouse cerebral hemodynamics was performed. We expect to realize new applications of photoacoustic and ultrasound imaging with new mouse models."

01

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Animal model

Figure 1 The creation process of the chronic cranial window (A-D) corresponds to steps 1~4.

Step 1

Mice were anesthetized by intraperitoneal injection of ketamine and toluene thiazide solution. Then lay the mouse flat on the operating table, and the operating table is heated to maintain the body temperature of the mouse. Use a scalpel to make a 10-minute incision on the scalp along the midline of the brain (Figure 1A).

Step 2

A pair of scalp fixers are used to separate the scalp along the incision. The meninges are torn along the sagittal suture. Then use a sterile swab to gently scrape off the meninges on the skull to maximize bone exposure. The skull drill is used to drill holes from the bottom of the coronary seam. The skull is gradually drilled to thin and groove along the coronary seam, and care must be taken to prevent the skull from being drilled through. Then, the drill bit moves down along the herringbone to perform the same drilling operation. After that, the drill bit moves along both sides of the skull to form a closed circular groove with a diameter of 10mm (Figure 1B).

Step 3

Gently drill a point hole at any point of the groove made in the previous step. Then use ophthalmological tweezers to remove the skull and expose the brain. Great care needs to be taken to avoid any damage to the meninges and cerebral cortex. Apply sterile cotton swabs repeatedly around the wound to prevent bleeding from the sub-skull capillary network (Figure 1C).

Step 4

Remove the scalp fixation, and suture the scalp after disinfection (Figure 1D). After craniotomy, 20% (volume fraction) of tofedine solution was injected subcutaneously, with a reference dose of 0.1 ml/20g body weight. This kind of surgery is to protect brain tissue from being scratched by wounds on the skull and scalp.

The mouse was kept alone in a dedicated cage to avoid accidental injury. The wound healed completely in 3 days. After 7 days of continuous recovery, the stitches were removed, and the mice were ready for imaging experiments. Each mouse with only a cranial window was imaged as a control with another normal mouse of the same age and weight. Please note that the treated mice are the same as normal mice in terms of experimental treatment. The cranial window is permanently created and protected by the scalp, and will not interfere with the activity and brain function of mice.

02

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PACT internal imaging

Figure 2A shows a simplified description of the COMPACT system and the imaging position/viewing angle.

The schematic diagram and imaging position of the USI system are shown in Figure 2B.

The schematic diagram of the photoacoustic mesoscopic mirror device is shown in Figure 2C.

Fig. 2 Schematic of the imaging system. (A) Top view of the circular array PACT (photoacoustic) system and mouse head. The dotted line marks the coronal plane position for imaging. tr: transducer array, fb: fiber bundle, fr: focusing region. (B) Top view of the USI (ultrasound) system setup and mouse head. The dotted line marks the coronal plane position for imaging. TR: transducer probe, AF: acoustic focal plane. (C) Photoacoustic mesoscopic system and three views.

03

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Verification of animal safety and health status

Representative images of the midbrain and hindbrain are shown in Figure 3A. Although slight cortical protrusion was observed within the cranial window, the structure of the entire brain remained normal. The mice were then euthanized for histopathological examination, and thin slices of the brain were stained with hematoxylin and eosin (H&E) and observed under a microscope. Comparison of the model mice with the control group is shown in Figure 3B.

Figure 3 MRI and histopathologic results. (A) MRI images of normal mice (top row) and model mice (bottom row), with the left column being the midbrain and the right column being the hindbrain. (B) H&E-stained images of different regions of the midbrain coronal plane.

04

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PACT imaging results

The effectiveness of the model was verified by the COMPACT imaging experiment. From the frontal lobe to the midbrain, take 20 coronal slices with a step size of 250mm. An enlarged view of the cranial cavity shows 3 typical layers (Figure 4).

Figure 4 PACT imaging results. (A) Planar PACT images, magnified for normal mice (left column) and mouse model (right column). (B) Planar images of RRV, lateral nasal vein, AchA, and anterior choroidal artery. (C) Planar images: LSCA, lateral superior cerebellar artery. (D) Vessel profiles with different color markings in PACT images.

05

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Ultrasound imaging and PA-US bimodal imaging results

We obtained coronal and sagittal ultrasound images (Figure 5).

Figure 5 USI imaging results. (A) Gray-scale image of a normal mouse, from left to right in the plane shown in Figure 2B (A - C). (B) Gray-scale image of a mouse model in the same plane as (A). (C) Doppler image of a normal mouse. Gray-scale images are shown on the left. Two different layers are shown in the top and bottom rows. (D) Doppler image of a mouse model in the same plane as (C).

Figure 6 shows the PACT images superimposed on the corresponding grayscale ultrasound images in the three representative planes. By comparing the images with those of normal mice, a significant increase in feature richness can be observed.

Fig. 6 PA-US dual-mode imaging results. the PA images are in pseudo-color and the US images are in grayscale as background. (A) PA-US images of mouse model in three coronal planes. (B) PA-US image of a normal mouse in the same plane as in (A).

06

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Photoacoustic microimaging results

The three-dimensional whole brain imaging results of the mouse model and normal mice were reconstructed and compared. Each situation provides a different perspective of observation (Figure 7)

Figure 7. photoacoustic microscope system and imaging results. (A) The whole brain imaging results of the MOUSE model. From top to bottom are the maximum intensity projections of the viewing angle shown in Figure 2C. (B) Imaging results of normal mice.

07

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PACT imaging findings in functional cerebral hemodynamics

The PA imaging results showed that the brain areas that showed enhanced signal after stimulation were in good agreement with the cerebrovascular (figures 8A and B), proving that the detected functional signals were caused by hemodynamic changes.

Figure 8 Functional cerebral hemodynamic PACT imaging results. Functional hemodynamic changes are displayed in pseudo-color, and the structural image is based on grayscale as the background. (A) No EXTERNAL STIMULATION (LEFT), LEFT front paw STIMULATION (MIDDLE), AND RIGHT FRONT paw STIMULATION (RIGHT) corresponding TO THE midbrain functional PACT IMAGE. (B) FUNCTIONAL PACT IMAGES OF the HIND brains OF THE THREE CASES shown IN FIGURE (A). (C) The RELATIVE CHANGES in THE TOTAL SIGNAL of the cerebral cortex in the brain under three different conditions shown IN figure (A). (D) The figure of the back of the head is the same as figure (C).

08

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Summary

We have successfully applied this technique to various classes of laboratory mice, including nude mice (CD-1 nude, Balb/c nude) and normal laboratory mice (CD-1, Balb/c). Currently, the cranial window mainly covers the top of the mouse skull. It would be preferable if the window could be extended to partially open the sides of the skull. However, this is expected to disrupt normal activity and pose a health risk. Further research is needed to determine the size limit of the cranial window while ensuring biosafety.

DOI：10.3389/fnins.2021.673740.

source (of information etc)：Frontiers in Neuroscience,2021, 15:
673740.

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