脳活動のメソスケール広視野光学イメージングのために大きな横開頭術手順

The overall goal of this large lateral craniotomy is to perform real time imaging over an expansive area of a cortical hemisphere. The main advantage of this technique is that large areas of the cortex are exposed for observation and can be used in conjunction with technologies such as optogenetics. This technique can provide insights into characterizing the neural dynamics between cells as well as that between different brain circuits at different temporal and spatial resolutions.

Visual demonstration of this method is critical due to the increased risk of hemmorage and cortical damage since the dura is often attached to brain surface and blood vessels. This method can be applied to study the neurobiological mechanisms underlying brain circuit development and its changes throughout life in response to new experiences or pathologies such as Alzheimer's Disease or stroke. To begin this procedure, autoclave all the surgical supplies and ensure that sterility is maintained throughout the surgery.

Ensure there is enough brain buffer for the surgery. Next, transfer an anesthetized mouse to the head holder setup and place it on a thermo-regulating heating pad set to 37 degrees Celsius. Secure the mouse's upper teeth in a teeth holder.

Then rotate its head towards the left approximately 30 degrees to expose the right lateral side of the head. Subsequently, secure the mouse's head with the blunt end of the ear bars. Then apply a thalmic ointment to prevent corneal drying.

To reduce cerebral edema, inject it with dexamethasone at four milligrams per kilogram intermuscularly. Wipe the skin over the surgical area with cotton swabs dipped in 4%chlorhexidine three times and then with 70%alcohol three times. Inject lidocaine subcutaneously over the craniotomy site.

Under a dissecting microscope, lift up the skin at one millimeter left of the midline just behind the ear with forceps and make a small horizontal incision with surgical scissors. Make a five to six millimeter lateral cut towards the right ear and then cut towards the rostral end of the head. At the initial incision point, insert the scissors and cut 10 millimeters rostrally.

After that, cut the skin around the right ear and near the right eye to expose the right side of the skull and temporal muscle. Ensure that the widest part of the exposed area is at least seven millimeters. Then trim the skin further if the surgical area needs to be extended.

Using a cotton swab, rub the surface of the skull in a circular motion to remove the periosteum from the skull. Ensure that no periosteum remains and that the skull is completely dry, which is critical for successful attachment of the head plate. Next, using spring scissors and forceps, separate the temporal muscle from the skull.

Cut and retract the muscle laterally until it reaches the squamosal bone. Take extreme care not to damage the superficial temporal vein that runs along the level of the squamosal bone near the eye, otherwise hemorrhage might occur. Control bleeding with gel foam pre-soaked in brain buffer.

For serious hemorrhage, use a heat cauterizer. Drop butyl cyanoacrylate glue onto bleeding sites. To begin this procedure, first mark bregma to orient the underlying brain regions, apply ethyl cyanoacrylate glue around the bottom edge of the head plate and glue the head plate over the craniotomy area.

After that, fill the opening space between the skull and the head plate with dental cement, leaving only the craniotomy area exposed. Then wait for the dental cement to dry and harden. Subsequently, outline the cranial window region by lightly scoring the surface of the skull with the dental drill.

Gently trace the drill along the original scoring to deepen it, ensuring that the drill does not penetrate through the skull into the brain. In the meantime, switch between drilling and dabbing the skull's surface with moistened rolled tissues every few minutes. This very challenging start requires surgeons to drill through the aged skull to free it for removal without damaging the underlying cortex.

Slow is loose, smooth is fast. During drilling, periodically check for buckling of the skull by gently pressing on it with forceps or the non-moving drill bit. When the bone begins the buckle, stop drilling and immerse the entire window in brain buffer.

Wait for at least five minutes before skull removal to soften the bone and to reduce the chance of the dura sticking to the bone, making the skull removal process easier. Next, perform the skull removal process beginning at the anterior edge. Gently pry the loose skull from the dura using forceps.

Once the bone is loose and floating on the dura, firmly grasp it with forceps and lift it away from the dura. Ensure the bone never penetrates into the brain. Use forceps and spring scissors to gently tear and cut away the dura.

Care must be taken to ensure the brain parenchyma and blood vessels are not damaged. Continue until all dura is removed from the cranial window site. When performed correctly, the brain will appear to be very clean with distinct blood vessels and no blemishes.

Spray the coverglass with 70%ethanol and use an air canister to gently dry. Ensure the glass is completely clean with no spots or dust present. Next, prepare 1.3%agarose by heating 200 milligrams of agarose powder dissolved in 15 milliliters of brain buffer.

Place a thermometer in the hot agarose and cool the agarose down to just above solidifying temperature at about 40 degrees Celsius. Cooling can be expedited by running cold water over the tube, taking care not to allow any water to enter the agar. Gently stir with the thermometer while monitoring its temperature.

Immediately before applying the agar, remove the brain buffer from the cranial well. Ensure no bubbles or particles are present as these will interfere with imaging. Now draw up the agarose with a transfer pipe head and drop the agarose directly on the brain.

Then quickly place the coverslip over the surface and fasten the coverslip with the agarose drops on the corners. Shown here is a photo micrograph of the wide unilateral craniotomy with bregma indicated by a white circle in each image. Patterns of cortical activation are shown in a mouse anesthetized with 0.5%isoflurane after stimulation of the contralateral whisker, auditory stimulation, contralateral forelimb stimulation, contralateral hindlimb stimulation and visual stimulation of the contralateral eye with a light-emitting diode.

There was midline activation from all forms of sensory stimulation at 10 to 25 milliseconds after primary sensory cortex activation. Once mastered, this technique can be completed in three to four hours. When attempting this procedure, it's important to remember to keep the blood loss to minimum and to ensure the cortex is not damaged.

Using this procedure, other imaging techniques such as optical imaging can help to answer important questions such as how neural dynamics are influenced by spontaneous activity in the normal and diseased brain. Following its development, this technique helped to pave the way for researchers in the field of systems neuroscience to explore interactions and neural dynamics between widely spaced cortical regions in mice. This video shows you how to perform a large lateral craniotomy in the mouse with minimal blood loss and cortical damage, enabling you to collect the necessary data to answer your most pressing scientific questions.