# A Bilateral Craniectomy Technique for In Vivo Photoacoustic Brain Imaging
## 3 Discussion  
The rat's skull, like the human skull, is a dense and acoustically heterogeneous structure. When photoacoustic waves pass through the skull, they can undergo scattering, reflection, and refraction (altogether referred to as skull aberration) due to the varying acoustic properties of the skull material. These aberrations can distort the photoacoustic signals and, in turn, can result in blurring, misalignment, or other artifacts in the reconstructed images and eventually make it more challenging to accurately visualize and locate specific brain structures or abnormalities. Many existing craniotomy methods are restricted because of their ability to provide only a limited view of the brain, typically allowing for unilateral exposure due to the presence of the midline superior sagittal sinus. More recent approaches have been developed that involve thinning or removing a section of the bone flap and replacing it with a transparent cover, enabling extended observation of the brain. Nonetheless, these craniotomies still offer relatively limited access. In this study, we presented a feasible approach for a novel bilateral hemicraniectomy technique in rat models, facilitating comprehensive exposure of the rat brain in vivo. Three primary goals were outlined prior to developing this method: first, maintain a bloodless field to minimize interference of the blood product with the imaging modality; second, preserve normal brain tissue architecture during the craniotomy; and third, create a large cranial window of approximately 1.5 cm to produce an adequate window for the device. In the development of this method, several techniques were attempted to satisfy these goals. One alternative method involved producing troughs with the drill to create a circular cranial flap to subsequently remove en bloc [1]. However, the dura was not consistently preserved in this process, and injury to the cortical surface near the troughs was observed. The sequential thinning process described above resulted in the least trauma to the cortical surface and the most reliable results, and by fully removing the bone over the bilateral cortices but keeping the dura intact, brain injury was minimized not only during the drilling but also during the photoacoustic imaging conducted in this study. While the dura could be removed delicately, it was purposefully kept intact in this method to protect the cortex during brain imaging; as the platform for photoacoustic imaging is lowered, it can be traumatic itself, and the dura helped reduce this injury. Additionally, in this method, to maximize visualization, bilateral exposure was required, but crossing the superior sagittal sinus posed a challenge. Fully removing the bone over the sinus invariably resulted in trauma to this structure, whether minor or major. Thus, the outer table of bone overlying the sinus, which corresponded to the sagittal suture, was removed, but importantly, the inner table of bone was kept intact in this thin strip. The technique described here differs considerably to other cranial exposures in the literature. In models of traumatic brain injury in the rat, both controlled cortical impact and water percussion injury models involve the use of a trephine to produce a 4-5 mm craniectomy, through which the injury is produced. In the water percussion injury model, midline and lateral craniotomies have been illustrated, whereby the dura is exposed, and then a female Lueer lock is cemented in place, allowing for the delivery of a pressure pulse from a column of fluid to induce trauma [8; 9; 10; 11]. In the controlled cortical impact model, the mechanism of injury differs: a piston generates the traumatic injury through direct mechanical force on an exposed brain [11]. These models suit the purpose of creating a cranial window to deliver the injury to a focal portion of the cortex, protecting the surrounding brain with an intact cranial window and allowing for animal survival for later experiments.