The overall goal of this procedure is to create a thin skull cranial window in a mouse model for in vivo optical coherence tomography imaging of the brain. This is accomplished by first anesthetizing the animal and preparing it for surgery. Next, thin the skull using various dental burrs.
The final step is to obtain OCT images of the brain in vivo through the thin skull cranial window. Ultimately, brain structural and functional information can be obtained from the resulting OCT images. The main advantage of this technique over existing methods such as craniectomy or cover slip windows, is that finical window is less invasive and more likely to maintain the biological integrity and Viability of the brain.
The implications of this technique extend towards the diagnosis of neurological disorders because OCT provides high spatial and temporal resolution and provides superior depth penetration when compared to confocal and two photon microscopy. Generally, individuals new to this method will struggle to create a uniformly thin skull surface without introducing skull fractures or subdural bleeding. To begin this procedure, anesthetize a CD one mouse between the ages of six to eight weeks with an intraperitoneal injection of ketamine and xylazine combination.
Then place the mouse on a homeo themic pad or blanket to ensure the optimal body temperature at approximately 37 degrees Celsius during the surgical preparation, continuously monitor the level of anesthesia by testing the animal's reflexes and inject more anesthesia when necessary. Next, lubricate both eyes with the artificial tear ointment. Remove the hair on the scalp with the clippers.
Then remove the residual clipped hair with a wedded pad or swab. After that, apply a thin layer of depilatory cream over the scalp. After two minutes, gently wipe away the cream and the remaining hair using saline, moistened cotton swabs and alcohol prep pads.
Now disinfect the scalp using a Betadine swab stick and clean with 70%ethanol prep pads. Then mount the animal onto a stereotaxic frame to immobilize the skull. Lightly tap the skull to ensure its stability.
In this procedure, start the scalp incision at the midline point between the eyes. Continue coddly to the midline point between the ears part, the skin flaps with forceps. Next, locate the area to be thinned under a dissecting microscope.
Use forceps to scrape the skull to remove any residual fascia. Then dry the skull with sterile cotton swabs before creating a four by four millimeter thinned cortical window about one millimeter posterior and lateral torema. Use a surgical hand drill with a round carbide burr in a light sweeping motion to thin the skull.
Do not apply direct pressure to the skull. Stop drilling every 20 to 30 seconds to remove bone dust using sterile saline and cotton swabs, and to avoid overheating the skull. The saline will also aid in dissipating the heat throughout the skull.
Once the outer layer of the compact bone is completely removed, the middle spongy bone layer should be visible. There may be slight bleeding as blood vessels are more apparent in the spongy bone layer. Switch to a green stone burr and continue drilling using extra caution.
As the spongy layer is more delicate, the green stone burr will remove less bone material while insuring evenness throughout the cranial window. Stop drilling occasionally to remove bone dust and to cool the skull. Finally, when the skull has become more transparent and the vasculature on the brain is visible, begin to polish the skull using a polishing bur check the thickness of the skull by gently tapping on it with forceps stop polishing.
When the skull becomes slightly flexible, the thinned cranial window should now be completely smooth and reflective and ready for imaging. Due to the nature of highly scattering tissues of the brain, the skull should be thinned to at least 55 microns for optimal depth penetration. After the surgery is complete, check the animal's breathing rate via visual inspection and reflexes to ensure proper level of anesthesia and administer additional anesthesia If necessary, place the animal under the OCT camera, then position the thin skull cranial window under the optical beam.
A cross sectional view of the skull and brain can now be visualized. Data acquisition can begin. Once the area of interest is located for imaging purposes, we scanned the OCT beam over an imaging cortical window with a width of 4.0 millimeters and obtain an imaging depth of two millimeters with six milliwatts of incident power and a focal 0.1 millimeter below the thin skull.
Each cross-sectional area consisted of 2048 axial scans with an acquisition rate of 0.14 seconds per image. This particular method has allowed us to identify specific structures in our OCT cross-sectional images. This figure shows a para sagittal OCT image of the cortex under a normal skull, and here shows a para sagittal OCT image under a thin skull.
The structures of the brain are more visually apparent under a thin skull cranial window as opposed to a normal skull. Additionally, a coronal cross-sectional OCT image is also obtained to facilitate in identifying midline structures. Volumetric scans of the brain can also be obtained by collecting a series of 2D cross-sectional images by using two sets of gal O mirrors for XY scanning with the first gal O mirror scanning the beam in the sagittal direction, and the second gal mirror scanning the coronal direction.
Once mastered, this technique can be completed in 10 minutes If it is performed properly. While attempting this procedure is important to uniformly thin the skull to ensure adequate signal penetration during OCT imaging, After watching this video, you should have a good understanding of how to prepare a thin skull cortical window for OCT imaging purposes.
