てんかんのマウス ・ モデルにおける Neurocardiac 機能障害を識別するために監視同時ビデオ脳波心電図

The overall goal of this procedure is to measure brain and heart biosignals in mice using simultaneous recordings of video, electroencephalography, and electrocardiography. This method can help answer key questions in the field of epilepsy, such as does a gene mutation in mice cause spontaneous seizures and do seizures evoke cardiac arrhythmias. The main advantage of this technique is that the tethered recording configuration allows more biopotential recording channels than wireless systems and it utilizes electrodes that are relatively inexpensive and disposable.

So this method can provide insight in cardiac dysfunction in epilepsy, it can also be applied to the study of neurocardiology to identify associations between arrhythmias and abnormal brain activity. To begin this procedure place the 10 socket female nano connector into a tabletop vice with the 10 wires facing up and the black wire in the front. Using fine forceps fold down the black wire to the right and the tan wire to the left.

Next fold down the red, orange, blue, and purple wires alternating right and left. Afterward, cut off the yellow, green, white, and gray wires at the base of their attachment. To prepare the ECG wires, use a permanent marker to make marks on the purple wire at 3.2 centimeters and 3.5 centimeters from the base of the electrode and on the blue wire at 2.2 centimeters and 2.5 centimeters.

Remove the electrode from the vice and expose the silver filaments between the marked areas by stripping the insulation on side of the wire with a scalpel blade. Subsequently, place the electrode back in the vice. Affix a piece of double sided mounting tape, which has be precut to the length and width of the electrode to the top of the wires using a thin layer of superglue.

Then trim the wires to be used for EEG at a slightly V-shaped angle to a length of approximately seven to nine millimeters, with the tan and black wires cut to the shortest. Keep in mind not to cut the wires to be used for ECG. In this procedure, shave two small areas about two square centimeters on both sides of the trunk of the mouse corresponding to where the ECG wires will be implanted.

Next, place the mouse in the prone position on the stage of the dissecting microscope and confirm the adequate depth of anesthesia by the absence of the toe pinch reflex. Holding the head steady between the thumb and forefinger, part the fur down the center of the head from between the ears to just behind the eyes with a cotton swab soaked I alcohol. Using a scalpel make a one centimeter midline incision through the scalp between the parted fur, from just in front of the ears to the just between the eyes.

Using either the side of the scalpel or a cotton tip applicator, gently scrape the mucus membrane on top of the skull until the bone appears dry. Then pluck the fur around the perimeter of the incision forming a thin border of bald skin. Carefully remove any fur that may have fallen into the surgical field with a pair of forceps.

Dry the surface of the skull with a cotton tip applicator applying gentle pressure for several seconds if needed. Following that, make four marks on the skull with a permanent marker at the sites where the burr holes will be drilled. Place two marks, one on each side of the sagittal suture, anterior to bregma for the reference and ground wires.

Subsequently place another two marks, one one each side of the sagittal suture posterior to bregma for the two EEG recording wires. Using a microdrill, make small burr holes at each mark with a 0.8 millimeter diameter drill bit. Apply gentle pressure while drilling to create small recesses at each marked spot.

Drill through the skull by pulsing the drill bit as the hole nears completion, being sure not to apply too much pressure, which could lead to penetrating and damaging the underlying brain tissue. After all holes are drilled, wipe the area clean with a cotton tip applicator. To adhere the electrode to the top of the skull, remove the paper backing from the double sided mounting tape on the electrode.

Apply a thin layer of superglue to the tape. Using a pair of forceps, remove the electrode from the vice. Orient the electrode such that when positioned along the sagittal suture, the shorter EEG wires are rostral and the longer ECG wires are caudal.

Adhere the electrode to the skull over the sagittal suture between the holes. To implant the wires for ECG, rotate the mouse slightly onto its right side, while keeping the head upright. Take the long ECG wire on the left side and extend it down the side of the mouse to the shaved area on the left side.

Visualize where the exposed wire will be positioned once it is tunneled beneath the skin. Then using a scalpel, make a on centimeter incision in the skin at the location where the exposed wire will be positioned. While holding the incision open, use forceps to loosen the skin around the incision from the underlying connective tissue to form a pocket for the wire.

Beginning at the incision site on the side of the animal tunnel subcutaneously with a piece of six centimeter long polyethylene tubing with a beveled leading edge until the beveled edge exits the incision made on the head. Feed the ECG wire through the tubing. While removing the tubing, grasp the electrode wire as it exits the lateral incision and pull the wire taut.

Next, fix the ECG wire in place by suturing it to the tissue under the skin. Apply one suture over the exposed filaments and another suture either before or after the exposed portion. Cut the electrode wire about two to three millimeters past the last suture and tuck the end into to the pocket of skin formed previously.

After that, pull two sides of the incision together and close with a wound clip. Repeat the above steps to place the contralateral ECG wire. To implant the wires for EEG, starting with the most anterior hole on one side, bend the wire that is closest to that hole so that it is positioned directly over the hole, but not yet inserted.

Grasp the lower end of the wire and feed it as horizontally as possible into the hole until two to three millimeters of the wire is under the skull. With the end of the wire secured in the hole gently fold down the remaining portion of the wire so that it lies flat against the skull. Continue in the same manner with the posterior wire on the same side.

Repeat the procedures for the anterior and posterior wires on the other side. In this procedure, mix two scoops of polycarboxylate powder with five drops of polycarboxylate liquid. Stir the mixture with a toothpick to make a paste with the desired viscosity.

Then pick up a large drop of cement with the toothpick and apply around the base of the electrode beginning caudally. Continue around the electrode allowing the cement to drip over the wires, forming a cap around the implant. Using to Dumont forceps, pull the fur at the edges of the incision up over the cement cap and press together, being careful not to disturb the wires implanted beneath.

Subsequently press the fur up into the cement to help with closure. After a recovery period, tether the mouse by gently but firmly holding the mouse in one hand while using the other hand to insert the 10 pin metal connector with guidepost into the sockets of the EEG, ECG electrode implant on the mouse's head. Then transfer the mouse to a recording chamber with transparent walls to facilitate video monitoring.

Record simultaneous video and EEG, ECG and save the digitized data for offline analyses with signal processing software. Shown here is an EEG trace representing spontaneous seizure in a KCNA1 knockout mouse. This is a plot of the time durations of each seizure observed during the 24 hour recording session in the mouse.

And this spectrogram shows the frequency and power density before, during, and after a seizure. Comparison of the relative power in each EEG frequency band during the pre and post-ictal periods reveals an increase in relative delta power and decreases in theta, alpha, beta, and gamma power. And this is a representative plot of an R-R interval series obtained from the ECG recording of the KCNA1 knockout mouse, showing the fluctuations in the time between beats.

The red line shows the low frequency trend components that give removed from the R-R interval series following detrending. Here is a sample ECG trace from a KCNA1 knockout mouse showing normal sinus rhythm preceding an atrioventricular conduction block, which manifests as a P-wave that is not followed by QRS complex. Once mastered, this surgery can be done in 50 minutes if it is performed properly.

After watching this video, you should have a good understanding of how to implant electrodes for recording electroencephalography and electrocardiography in mice, which can be applied in epilepsy models to identify neurocardiac dysfunction.