We acknowledge the following funding sources: NIH NINDS &#8211; K08NS101122 (JB), R01NS040337 (JK), R01NS044370 (JK), University of Virginia School of Medicine (JB).

All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Virginia.
1. Electrode building/cable building
<ol>
	<li>Use a unipolar insulated stainless-steel wire (0.005&#8221; bare diameter, 0.008&#8221; coated) to make an electrode that is connected with a female socket connector (female receptacle connector 0.079).</li>
	<li>Use a special custom-made cable to connect animals to the amplifier.
	<ol>
		<li>Attach a male 4-pin connector (M...

<ol>
	<li>Vasudevan, C., Levene, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+aetiology+of+neonatal+seizures.">Epidemiology and aetiology of neonatal seizures.</a> Seminars in Fetal &amp; Neonatal Medicine. , (2013).</li><li>Volpe, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+Seizures.">Neonatal Seizures.</a> Volpe's Neurology of the Newborn. , 275-321 (2018).</li><li>Shankaran, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+EKSNNR.+Childhood+outcomes+after+hypothermia+for+neonatal+encephalopathy.">Network EKSNNR. Childhood outcomes after hypothermia for neonatal encephalopathy.</a> New England Journal of Medicine. 366 (22), 2085-2092 (2012).</li><li>Pappas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+outcomes+after+neonatal+encephalopathy.">Cognitive outcomes after neonatal encephalopathy.</a> Pediatrics. 135 (3), 624-634 (2015).</li><li>van Schie, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+motor+and+behavioral+outcome+after+perinatal+hypoxic-ischemic+encephalopathy.">Long-term motor and behavioral outcome after perinatal hypoxic-ischemic encephalopathy.</a> European Journal of Paediatric Neurology. 19 (3), 354-359 (2015).</li><li>Rensing, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+analysis+of+developmental+changes+in+electroencephalography+patterns+and+sleep-wake+states+of+the+neonatal+mouse.">Longitudinal analysis of developmental changes in electroencephalography patterns and sleep-wake states of the neonatal mouse.</a> PLoS One. 13 (11), 1-17 (2018).</li><li>Rice, J. E., Vannucci, R. C., Brierley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+immaturity+on+hypoxic-ischemic+brain+damage+in+the+rat.">The influence of immaturity on hypoxic-ischemic brain damage in the rat.</a> Annals of Neurology. 9 (2), 131-141 (1981).</li><li>Burnsed, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-ischemia+and+therapeutic+hypothermia+in+the+neonatal+mouse+brain--a+longitudinal+study.">Hypoxia-ischemia and therapeutic hypothermia in the neonatal mouse brain--a longitudinal study.</a> PLoS One. 10 (3), 0118889 (2015).</li><li>Semple, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+development+in+rodents+and+humans:+Identifying+benchmarks+of+maturation+and+vulnerability+to+injury+across+species.">Brain development in rodents and humans: Identifying benchmarks of maturation and vulnerability to injury across species.</a> Progress in Neurobiology. , 1-16 (2013).</li><li>Comi, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gabapentin+neuroprotection+and+seizure+suppression+in+immature+mouse+brain+ischemia.">Gabapentin neuroprotection and seizure suppression in immature mouse brain ischemia.</a> Pediatric Research. 64 (1), 81-85 (2008).</li><li>Comi, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+stroke+and+ischemic+seizures+in+the+immature+mouse.">A new model of stroke and ischemic seizures in the immature mouse.</a> Pediatric Neurology. 31 (4), 254-257 (2004).</li><li>Kadam, S. D., White, A. M., Staley, K. J., Dudek, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+Electroencephalographic+Monitoring+with+Radio-Telemetry+in+a+Rat+Model+of+Perinatal+Hypoxia-Ischemia+Reveals+Progressive+Post-Stroke+Epilepsy.">Continuous Electroencephalographic Monitoring with Radio-Telemetry in a Rat Model of Perinatal Hypoxia-Ischemia Reveals Progressive Post-Stroke Epilepsy.</a> Journal of Neuroscience. 30 (1), 404-415 (2010).</li><li>Burnsed, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+Circuit+Activity+during+Neonatal+Hypoxic+-+Ischemic+Seizures+in+Mice.">Neuronal Circuit Activity during Neonatal Hypoxic - Ischemic Seizures in Mice.</a> Annals of Neurology. 86, 927-938 (2019).</li><li>Sampath, D., White, A. M., Raol, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+neonatal+seizures+in+an+animal+model+of+hypoxic-ischemic+encephalopathy.">Characterization of neonatal seizures in an animal model of hypoxic-ischemic encephalopathy.</a> Epilepsia. 55 (7), 985-993 (2014).</li><li>Sampath, D., Valdez, R., White, A. M., Raol, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticonvulsant+effect+of+flupirtine+in+an+animal+model+of+neonatal+hypoxic-ischemic+encephalopathy.">Anticonvulsant effect of flupirtine in an animal model of neonatal hypoxic-ischemic encephalopathy.</a> Neuropharmacology. 123, 126-135 (2017).</li><li>Kang, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=and+sex-dependent+susceptibility+to+phenobarbital-resistant+neonatal+seizures:+role+of+chloride+co-transporters.">and sex-dependent susceptibility to phenobarbital-resistant neonatal seizures: role of chloride co-transporters.</a> Frontiers in Cellular Neuroscience. 9, 1-16 (2015).</li><li>Zanelli, S., Goodkin, H. P., Kowalski, S., Kapur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+transient+acute+hypoxia+on+the+developing+mouse+EEG.">Impact of transient acute hypoxia on the developing mouse EEG.</a> Neurobiology of Disease. 68, 37-46 (2014).</li><li>Lewczuk, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EEG+and+behavior+patterns+during+experimental+status+epilepticus.">EEG and behavior patterns during experimental status epilepticus.</a> Epilepsia. 59 (2), 369-380 (2017).</li><li>Wu, D., Martin, L. J., Northington, F. J., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillating+gradient+diffusion+MRI+reveals+unique+microstructural+information+in+normal+and+hypoxia-ischemia+injured+mouse+brains.">Oscillating gradient diffusion MRI reveals unique microstructural information in normal and hypoxia-ischemia injured mouse brains.</a> Magnetic Resonance in Medicine. 72 (5), 1366-1374 (2014).</li></ol>

We have presented a model for continuous video-EEG monitoring in neonatal mice during hypoxic-ischemic seizures. Video analysis in conjunction with EEG allows characterization of seizure semiology. Analysis of EEG allows for extraction of power spectrograms and background amplitude analysis.
Correct and careful placement of electrodes is crucial in this protocol, as injury during electrode placement or inaccurate placement can significantly affect results. Assessment of normal baseline EEG act...

Hypoxic ischemic encephalopathy (HIE) is a condition that affects 1.5 in 1000 newborns annually and is the most common cause of neonatal seizures1,2. Infants who survive are at risk for various neurological disabilities such as cerebral palsy, intellectual disability, and epilepsy3,4,5.
Animal models play a critical role in understanding and investigating the pathophysiology of hypoxia ischemia and neonatal seizures6,7. A modified Vannucci model is used to induce hypoxia ischemia (HI) on postnatal day 10 (p10)7,8. Mouse pups of this age translate neurologically roughly to the full term human neonate9.
Continuous video electroencephalography (EEG) monitoring used in conjunction with this injury model allows for further understanding and characterization of neonatal hypoxic ischemic seizures. Previous studies have used various methods for analyzing neonatal seizures in rodents, including video recordings, limited EEG recordings and telemetry EEG recordings10,11,12,13,14,15,16. In the following manuscript, we discuss in depth the process of recording continuous video EEG in mouse pups during hypoxia-ischemia. This technique for continuous video EEG monitoring in neonatal mouse pups could be applied to a variety of disease and seizure models.

<table>
	<tbody>
		<tr>
			<td>SURGERY</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ball Point Applicator</td>
			<td>Metrex Research</td>
			<td>8300-F</td>
			<td>i-bond applicator</td>
		</tr>
		<tr>
			<td>Cranioplast (Powder/Resin)</td>
			<td>Coltene</td>
			<td>H00383</td>
			<td>Perm Reline/Power</td>
		</tr>
		<tr>
			<td>I-Bond</td>
			<td>Kulzer GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LOOK Silk Suture</td>
			<td>Surgical Specialities Corporation</td>
			<td>SP115</td>
			<td>LOOK SP115 Black Braided Silk Non absorbable surgical suture</td>
		</tr>
		<tr>
			<td>RS-5168 Botvin Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS5168</td>
			<td>Forcep for surgery/ligation</td>
		</tr>
		<tr>
			<td>RS-5138 Graefe Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS5138</td>
			<td>Forcep for surgery/ligation</td>
		</tr>
		<tr>
			<td>UV light for I-Bond</td>
			<td>Blast Lite By First Media</td>
			<td>BL778</td>
			<td>UV ligth for I-bond</td>
		</tr>
		<tr>
			<td>Vannas Microdissecting Scissor</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS5618</td>
			<td>Scissor for ligation</td>
		</tr>
		<tr>
			<td>Vet Bond</td>
			<td>3M Vetbond</td>
			<td>1469SB</td>
			<td>Vet Glue</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HYPOXIA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxidial</td>
			<td>Starr Life Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen sensor</td>
			<td>Medical Products</td>
			<td></td>
			<td>MiniOxI- oxygen analyzer/sensor for hypoxia rig</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EEG RECORDING</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Female receptacle connector 0.079&#34;</td>
			<td>Mill-Max Manufacturing Corp</td>
			<td>832-10-024-10-001000</td>
			<td>Ordered from Digikey</td>
		</tr>
		<tr>
			<td>Grass Amplifier</td>
			<td>Natus Neurology Incorporated</td>
			<td></td>
			<td>Grass Product</td>
		</tr>
		<tr>
			<td>LabChart Pro</td>
			<td>ADI Instruments</td>
			<td></td>
			<td>Software to run the system</td>
		</tr>
		<tr>
			<td>Male Socket Connector 0.079&#34;</td>
			<td>Mill-Max Manufacturing Corp</td>
			<td>833-43-024-20-001000</td>
			<td>Ordered from Digikey</td>
		</tr>
		<tr>
			<td>Operational Amplifier</td>
			<td>Texas Instruments, Dallas, TX, USA</td>
			<td>TLC2274CD</td>
			<td>TLC2274 Quad Low&#8208;Noise Rail&#8208;to Rail Operational Amplifier</td>
		</tr>
		<tr>
			<td>Operational Amplifier</td>
			<td>Texas Instruments, Dallas, TX, USA</td>
			<td>TLC2272ACDR</td>
			<td>TLC2274 Quad Low&#8208;Noise Rail&#8208;to Rail Operational Amplifier</td>
		</tr>
		<tr>
			<td>Stainless Steel wire</td>
			<td>A-M Systems</td>
			<td>791400</td>
			<td>0.005&#34; Bare/0.008&#34; Coated 100 ft</td>
		</tr>
		<tr>
			<td>Ultra-Flexible Wire</td>
			<td>McMaster-Carr</td>
			<td>9564T1</td>
			<td>36 Gauze wire of various color</td>
		</tr>
	</tbody>
</table>

continuous video electroencephalogram during hypoxia-ischemia in neonatal mice

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying neonatal seizures and hypoxia ischemia. This manuscript describes a method for continuous video electroencephalogram (EEG) monitoring in neonatal mice to detect seizures and analyze EEG background during hypoxia ischemia. Use of video and EEG in conjunction allows description of seizure semiology and confirmation of seizures. This method also allows analysis of power spectrograms and EEG background pattern trends over the experimental time period. In this hypoxia ischemia model, the method allows EEG recording prior to injury to obtain a normative baseline and during injury and recovery. Total monitoring time is limited by the inability to separate pups from the mother for longer than four hours. Although, we have used a model of hypoxic-ischemic seizures in this manuscript, this method for neonatal video EEG monitoring could be applied to diverse disease and seizure models in rodents.

Seizure semiology
Neonatal hypoxia-ischemia exposure results in both generalized and focal seizures in mice (Figure 1A-C). Video EEG recordings allow electrographic findings to be correlated to behavior on video. These behaviors were scored using a previously published neonatal rodent behavioral seizure score (BSS)16. In addition to BSS, we categorized events based on whether the behavior was focal/unilateral, bila...

Watch this Scientific Journal Video about Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice at JoVE.com

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

This manuscript describes a method for continuous video EEG recordings using multiple depth electrodes in neonatal mice undergoing hypoxia-ischemia.

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying ...

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.

continuous-video-electroencephalogram-during-hypoxia-ischemia

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JoVE Journal

Neuroscience

3.2K vistas.  University of Virginia. 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.Demons...