Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

Hypoxic ischemic encephalopathy is the most common cause of neonatal seizures. This mouse model of hypoxic ischemic seizures and continuous video electroencephalography or EEG can help further our understanding and characterization of neonatal seizures. This technique facilitates the acquisition of high quality video electroencephalography recordings in a neonatal injury model and can aid in future studies of the mechanisms of neonatal seizures, outcomes, and therapeutic testing.

Demonstrating the procedure will be Pravin Wagley. A lab specialist from my laboratory. For electrode implantation surgery.

After confirming a lack of response to pain reflex in an anesthetized postnatal day nine mouse, place the mouse in a stereotaxic stage with a nose cone and use the reverse side of the ear bar to hold the head steady. Sterilize the incision area on the skull with three alternating betaine and 70%ethanol scrubs and use a fenestrated drape to cover the mouse while keeping the incision region visible. Open the scalp anterior posterior from slightly above the eyes, and retract approximately half a centimeter of skin.

Reposition the head on the stereotaxic stage so that the skin pulls outward to expose the skull, and use a cotton swab to apply hydrogen peroxide. Use a scalpel blade to carefully scrape the skull clean. And apply an approximately 50 microliter drop of adhesive to the bone.

Use the applicator to spread the adhesive over the skull and place the adhesive under ultraviolet light for 40 seconds. When the adhesive has set, measure the coordinates using the exposed bregma as a reference. And use a 32 gauge needle to create electrode holes bilaterally in the CA1 region of the hippocampus, bilaterally in the parietal cortex and in the cerebellum.

Clean the blood from the surface of the skull and use the stereotaxic arm to lower the electrodes attached to the female socket connector into the brain. Implant each electrode into the brain and fix the electrodes in place with dental acrylic. Glue the headset to the skull with additional dental acrylic.

Subcutaneously inject five milligrams per kilogram of Ketoprofen into each pup. When all of the electrodes have been secured in at least half of the litter return the pups to the mother. 24 hours after electrode implantation, place each animal in a 37 degree Celsius custom made plexiglas chamber for the EEG recording and use a flexible custom made operational amplifier cable to connect the pups to a video EEG monitoring system.

Use a multi-channel differential amplifier to digitize the EEG data at 1000 Hertz with a one kilo gain and review the EEG signal in appropriate analysis software. Then record a pre-injury baseline EEG for 30 minutes, before disconnecting the animals for the carotid artery litigation procedure. After recording the baseline EEG data, sterilize the skin between the mandible and the clavicle on the left side of the neck with betaine and ethanol as demonstrated.

And make an approximately one centimeter incision on the left side of the neck. Place the pup under a dissecting microscope and carefully retract the subcutaneous tissue and skin to expose the carotid artery. Place a five centimeter piece of sterile silk suture under the artery.

Identify the vagus nerve and delicately separate and retract the nerve from the artery. Maneuver the suture so that it is only under the artery. After identifying the vagus nerve, delicately separate and retract the nerve from the artery and use micro forceps to thread a five centimeter piece of sterile silk suture under the artery.

Tie a double knotted suture around the artery to occlude the flow and cut the excess suture. Replace the subcutaneous tissue and skin over the exposed artery and use Vetbond to seal the incision. Then place the animal back on continuous EEG monitoring in a chamber at room temperature placed on warming mattress and take spot infrared temperature checks of the pup core temperature to avoid open the chamber.

After allowing the pups to recover for one hour with continuous fraction of inspired oxygen monitoring, flush the chamber with 60 liters per minute of 100%nitrogen and 0.415 liters per minute of 100%oxygen. Once the oxygen saturation in the chamber reaches 12%decrease the nitrogen flow to 10 liters per minute while keeping the oxygen flow unchanged. Making small adjustments as necessary, maintain the fraction of inspired oxygen at 8%for 45 minutes.

After 45 minutes of hypoxia exposure, return the fraction of inspired oxygen to 21%and allow the pups to recover in the chamber with EEG monitoring for two hours. At the end of the recording period, disconnect the mice from the EEG system and return the pups to the mother. Video EEG recordings allow electrographic findings to be correlated to behavior on video.

The majority of events observed in this model involve focal unilateral or mixed behaviors. During the hypoxic period, a subset of mice typically exhibit non-compulsive seizure activity, during which the pups are immobile with sustained seizure activity on EEG. In this analysis, the baseline activity was similar to the previously described background in P 10 mouse pups.

After litigation and before the commencement of hypoxia, a subset of mice exhibited convulsive seizures. Following hypoxia induction, the background amplitude on the EEG was reduced. And intermittent bursts of spike wave discharges were exhibited, followed by suppression.

The mice exhibited electrographic seizures which emerge from a suppressed background as rhythmic spike wave discharges that progressed to become more complex and frequent with polys spike waves. During hypoxia, power spectrogram analysis is notable for asymmetries between the ischemic and contralateral hemispheres. The ischemic hemisphere exhibits a burst suppression pattern, and the contralateral hemisphere exhibits a suppressed background.

During re-oxygenation and recovery a subset of mice continues to have seizures over the remainder of the recording period. The EEG background is suppressed compared to baseline following hypoxia with a gradual recovery observed during the post hypoxia recording period. When working with neonatal pups, precision is key.

In addition, special attention should be taken to place the pups back with the mother in groups after the surgical procedures. Video electroencephalography can be used in neonatal pups in conjunction with histopathology, imaging, and behavior to examine the impact of neonatal seizures on injure surgery and behavioral outcomes.