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 (Male connector 0.079&#8221;) to 4 channel unity gain impedance matching operational amplifier (op-amp). Attach a 10K resistor to the wires that connect to the 9 V battery. A ground wire not connected to the op-amp acts as the midpoint of the battery.</li>
		<li>Connect one end of the cable (AWG, 0.012&#8221; OD) to the op-amp and connect the other end of the cable to the amplifier.</li>
	</ol>
	</li>
</ol>
2. Electrode implantation surgery
<ol>
	<li>Anesthetize the pup (postnatal day 9) with 4-5% isoflurane in a downward flow hood. Prior to the start of the procedure, inject the pups with bupivacaine (0.02-0.05 mL, 0.25% subcutaneous local infiltration).</li>
	<li>Once the animal is immobile, transfer to a stereotactic stage with a nose cone. Use the reverse side of the ear bar as it is soft to hold the head steady. At this age, pups do not have a fully developed ear to use the pointed end of the ear bar.</li>
	<li>Turn down the flow of isoflurane and maintain it at 2.5-3%. Keep an eye on steady breathing of the pup throughout the surgery procedure. Pinch the tail to check pain response and then proceed to incision.</li>
	<li>Sterilize the incision area on the skull with betadine and alcohol (3 cycles of alternating iodine and 70% ethanol). Drape the surrounding body part such that the incision region is visible.</li>
	<li>Open the scalp anterior-posterior from slightly above the eyes and retract approximately 0.5&#160;cm of skin. Reposition the mouse head on the stereotaxic stage so that the skin pulls outward exposing skull.</li>
	<li>Apply hydrogen peroxide on the skull using a cotton swab and scrape the skull clean using a scalpel blade. The skull is very soft; exercise caution while scraping.</li>
	<li>Apply one drop (approximately 50 &#181;L) of adhesive and spread it around the exposed skull area using its applicator. Expose to UV light for 40 s to set the adhesive.</li>
	<li>Measure the coordinates using the exposed bregma as the reference. Implant electrodes bilaterally in the CA1 region of hippocampus [-3.5 mm Dorsal-Ventral (DV), &#177;2 mm Medial-Lateral (ML), -1.75 mm Deep (D)] and bilaterally in the parietal cortex [-1.22 mm DV, &#177;0.5 mm ML, -1 mm D] and a reference electrode in the cerebellum17. Use a 32 G needle to create a hole at the marked region.</li>
	<li>Clean the blood from the surface of the skull. Lower electrodes attached to the female socket connector into the brain with the help of the stereotaxic arm and fix in place with dental acrylic. Implant the electrode in the brain. The socket connector headset sits on top of the skull glued together by dental acrylic.</li>
	<li>Inject ketoprofen (5 mg/kg) subcutaneously in the interscapular region once the electrode is fixed. Place the pups back with the mother. 
	NOTE: Introduce half of the litter with the headset at once to the mother rather than introducing them one at a time. This will prevent from mother damaging the pup&#8217;s headset.</li>
</ol>
3. EEG setup and recording (baseline/pre-injury)
<ol>
	<li>After 24 h of recovery after electrode implantation, place each animal in a heated (37 &#176;C) custom-made Plexiglas chamber for EEG recording. This chamber will also serve as a hypoxia chamber.</li>
	<li>Connect pups in the chamber to a video-EEG monitoring system via a flexible cable (custom made op-amp cable). 
	NOTE: With the headset in place, the mice are freely mobile and do not exhibit any differences in behavior. Once attached to the electrode wires, the wires must be adjusted within the chamber tether in order to provide the right amount of slack so that the pup can freely move throughout the chamber.</li>
	<li>Digitize the EEG data at 1000 Hz with 1K gain using a grass amplifier. Review the EEG signal (band pass filter between 3-70 Hz) later using software (e.g., LabChart Pro).</li>
	<li>Record a pre-injury baseline EEG for 30 minutes prior to disconnecting animals for carotid artery ligation procedure.</li>
</ol>
4. Left carotid artery ligation
<ol>
	<li>Anesthetize the pup (postnatal day 10) with 4-5% isoflurane in a downward flow hood and place them on specially arranged setup on a waterbath pad. Position the animal supine and secure the forelimbs with paper tape.
	<ol>
		<li>Lower the flow of isoflurane to 2-3%. Pinch the tail for pain response and monitor breathing throughout procedure.</li>
	</ol>
	</li>
	<li>Sterilize the incision area (between mandible and the clavicle) on the left side of the neck with betadine and alcohol (3 cycles of alternating iodine and 70% ethanol).</li>
	<li>Make an approximately 1 cm long incision on the left side of the neck using microscissors. Using a dissecting microscope, carefully retract the subcutaneous tissue and skin to expose the carotid artery. Take care to identify the vagus nerve (running lateral to the artery) and delicately separate and retract it from the artery.</li>
	<li>Thread a 5 cm long sterile silk suture under the artery using microforceps. Tie a double knotted suture around the artery to occlude flow.</li>
	<li>Cut the excess suture and close the exposed artery by pulling back the subcutaneous tissue and skin. Use vet bond to seal the incision.</li>
	<li>Place the animal back on continuous EEG monitoring in a chamber at room temperature, which is placed on a warming mattress. Take spot infrared temperature checks of pup core temperature to avoid opening the chamber. Allow the animal to recover for 1 h prior to hypoxia.</li>
</ol>
5. EEG and hypoxia
<ol>
	<li>Continuously monitor FiO2 (fraction of inspired oxygen) within the chamber via an oxygen monitor.</li>
	<li>Flush the chamber with 60 L/min of 100% N2 and 0.415 L/min for 100% O2. Once the oxygen saturation in the chamber reaches 12%, decrease the N2 flow to 10 L/min while keeping the O2 flow unchanged. With small adjustments, maintain the FiO2 at 8% for 45 min.</li>
	<li>After 45 min of hypoxia exposure, return FiO2 to 21%.</li>
	<li>Have pups recover in chamber and monitor on EEG for 2 h post-hypoxia.</li>
	<li>After completion of recording period, disconnect mice from EEG recording and return to the mother.</li>
</ol>
6. EEG analysis
<ol>
	<li>Analyze the EEG file with video in LabChart Pro. Have a blinded researcher mark the EEG for seizures and background patterns17. Seizures are defined as an electrographic event lasting greater than 10 seconds with high frequency rhythmic sharp wave discharges (&#8805;3x baseline) with clear evolution17.</li>
	<li>Have a second blinded researcher review marked events at random for agreement.</li>
	<li>Review associated video for each marked electrographic event and analyze according to the neonatal rodent behavioral seizure score16. Briefly, this score ranges from 0-6 (immobility to severe tonic-clonic behavior). To further characterize seizure semiology, analyze behavior for laterality (multifocal/bilateral movements vs. focal/unilateral vs. mixed).</li>
	<li>Create a power spectrogram. Use a Fast Fourier Transform with a Cosine-Bell data window with a size of 1024 data points. Create a smooth x-axis in the spectrogram with the help of a window overlap of 87.5%. Express the power as &#181;V218.</li>
</ol>

<ol><li>Vasudevan, C., Levene, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidemiology+and+aetiology+of+neonatal+seizures%5BTitle%5D)+AND+%22Seminars+in+Fetal+%26+Neonatal+Medicine%22%5BJournal%5D)">Epidemiology and aetiology of neonatal seizures.</a> Seminars in Fetal & Neonatal Medicine. , (2013).</li>
<li>Volpe, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neonatal+Seizures%5BTitle%5D)+AND+%22Volpe%27s+Neurology+of+the+Newborn%22%5BJournal%5D)">Neonatal Seizures.</a> Volpe's Neurology of the Newborn. , Elsevier. 275-321 (2018).</li>
<li>Shankaran, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Network+EKSNNR.+Childhood+outcomes+after+hypothermia+for+neonatal+encephalopathy%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">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/pubmed?term=((Cognitive+outcomes+after+neonatal+encephalopathy%5BTitle%5D)+AND+%22Pediatrics%22%5BJournal%5D)">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/pubmed?term=((Long-term+motor+and+behavioral+outcome+after+perinatal+hypoxic-ischemic+encephalopathy%5BTitle%5D)+AND+%22European+Journal+of+Paediatric+Neurology%22%5BJournal%5D)">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/pubmed?term=((Longitudinal+analysis+of+developmental+changes+in+electroencephalography+patterns+and+sleep-wake+states+of+the+neonatal+mouse%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((The+influence+of+immaturity+on+hypoxic-ischemic+brain+damage+in+the+rat%5BTitle%5D)+AND+%22Annals+of+Neurology%22%5BJournal%5D)">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/pubmed?term=((Hypoxia-ischemia+and+therapeutic+hypothermia+in+the+neonatal+mouse+brain--a+longitudinal+study%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Brain+development+in+rodents+and+humans%3A+Identifying+benchmarks+of+maturation+and+vulnerability+to+injury+across+species%5BTitle%5D)+AND+%22Progress+in+Neurobiology%22%5BJournal%5D)">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/pubmed?term=((Gabapentin+neuroprotection+and+seizure+suppression+in+immature+mouse+brain+ischemia%5BTitle%5D)+AND+%22Pediatric+Research%22%5BJournal%5D)">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/pubmed?term=((A+new+model+of+stroke+and+ischemic+seizures+in+the+immature+mouse%5BTitle%5D)+AND+%22Pediatric+Neurology%22%5BJournal%5D)">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/pubmed?term=((Continuous+Electroencephalographic+Monitoring+with+Radio-Telemetry+in+a+Rat+Model+of+Perinatal+Hypoxia-Ischemia+Reveals+Progressive+Post-Stroke+Epilepsy%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Neuronal+Circuit+Activity+during+Neonatal+Hypoxic+-+Ischemic+Seizures+in+Mice%5BTitle%5D)+AND+%22Annals+of+Neurology%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+neonatal+seizures+in+an+animal+model+of+hypoxic-ischemic+encephalopathy%5BTitle%5D)+AND+%22Epilepsia%22%5BJournal%5D)">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/pubmed?term=((Anticonvulsant+effect+of+flupirtine+in+an+animal+model+of+neonatal+hypoxic-ischemic+encephalopathy%5BTitle%5D)+AND+%22Neuropharmacology%22%5BJournal%5D)">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/pubmed?term=((and+sex-dependent+susceptibility+to+phenobarbital-resistant+neonatal+seizures%3A+role+of+chloride+co-transporters%5BTitle%5D)+AND+%22Frontiers+in+Cellular+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Impact+of+transient+acute+hypoxia+on+the+developing+mouse+EEG%5BTitle%5D)+AND+%22Neurobiology+of+Disease%22%5BJournal%5D)">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/pubmed?term=((EEG+and+behavior+patterns+during+experimental+status+epilepticus%5BTitle%5D)+AND+%22Epilepsia%22%5BJournal%5D)">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/pubmed?term=((Oscillating+gradient+diffusion+MRI+reveals+unique+microstructural+information+in+normal+and+hypoxia-ischemia+injured+mouse+brains%5BTitle%5D)+AND+%22Magnetic+Resonance+in+Medicine%22%5BJournal%5D)">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 activity prior to injury is paramount, as bleeding or injury during electrode placement, while rare, can happen. Secondly, in order to confirm correct electrode placement, brains can be sectioned and examined for electrode tracks in the proper placement. In addition, failure to return pups to the mother in groups (individually) may result in electrode headsets being damaged or pups being killed or neglected by the mother.
One limitation of this method is the limit of spatial localization of depth electrode recordings in a small neonatal brain. This restricts the ability to localize specific seizure foci on EEG recordings. Another limitation in this model of hypoxia ischemia is the variability in seizure burden. Variability in lesion size and behavioral deficits in this rodent model of hypoxia ischemia has been well described previously7,8,19. Not surprisingly, this variability exists in seizure burden (both length of seizure events and number of seizure events). However, consistently, 100% of pups in this model exhibit seizures during hypoxia. Lastly, the amount of time pups can be on EEG monitoring (away from the mother) is limited. Therefore, we are unable to characterize ongoing seizures with continuous EEG at later time points relative to the injury.
Although, we have used a hypoxia-ischemia seizure model in this manuscript, this method for continuous video-EEG monitoring in neonatal mouse pups could be easily applied to other disease/seizure models. Seizures in neonatal rodents are difficult to recognize based on behavior alone, making video-EEG monitoring important. Future investigations could use these techniques to analysis seizure burden and semiology in other neonatal seizure models or response to therapeutics and neuroprotective measures.

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>&lt;strong&gt;SURGERY&lt;/strong&gt;</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>&lt;strong&gt;HYPOXIA&lt;/strong&gt;</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>&lt;strong&gt;EEG RECORDING&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Female receptacle connector 0.079&quot;</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&quot;</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-Noise Rail-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-Noise Rail-to Rail Operational Amplifier</td></tr><tr><td>Stainless Steel wire</td><td>A-M Systems</td><td>791400</td><td>0.005&quot; Bare/0.008&quot; 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, bilateral, or mixed (Figure 1B).
In this model, mice generally exhibited 3 patterns of seizure semiology: 1) repetitive circling to the side of ligation with extension of contralateral extremities, 2) loss of posture with body flexion and tail curled to side of ligation, or 3) loss of posture with unilateral or bilateral paddling of extremities (varying severity and length). The majority of observed events involved focal/unilateral or mixed behaviors (Figure 1B). In addition, during the hypoxic period, a subset of mice exhibited non-convulsive seizure activity, where the pup was immobile with sustained seizure activity on EEG (Figure 1C).
Electrographic recordings
EEG recording was started 30 minutes prior to carotid ligation in order to obtain a pre-injury baseline. Baseline activity (Figure 1A and Figure 2A) was similar to previously described background in p10 mouse pups17. Following ligation, pups were immediately placed back on video EEG. During the period between ligation and commencement of hypoxia, a subset of mice exhibits convulsive seizures (Figure 1A-C).
Following hypoxia induction, background amplitude on EEG reduced (Figure 3B) and intermittently exhibited bursts of spike-wave discharges, followed by suppression (Figure 2A). Mice exhibit electrographic seizures, which emerge from a suppressed background as rhythmic spike-wave discharges and progress to become more complex and frequent, with polyspike waves (Figure 2B). During hypoxia, power spectrogram analysis was notable for asymmetries between the ischemic and contralateral hemisphere (Figure 3A,B). The ischemic hemisphere exhibited a burst suppression pattern and the contralateral hemisphere exhibited suppressed background (Figure 1A and Figure 3A,B). On average seizures begin 5.5&#177;8.1 minutes after induction of hypoxia, with each event lasting 56&#177;57 seconds. There was a 13% mortality rate during hypoxia (n=4/30), with all deaths following a convulsive (BSS=5-6) seizure.
During reoxygenation and recovery, a subset of mice continues to have seizures over the remainder of the recording period (2 h post-hypoxia). EEG background was suppressed compared to baseline following hypoxia (Figure 1A and Figure 3), with gradual recovery during the post-hypoxia recording period. Over the entire recording period, mice exhibited on average 9&#177;5 seizure events, each lasted 54&#177;57.7 s.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61346/61346fig1v2.jpg" /> 
Figure 1: Seizure characteristics in p10 mice exposed to neonatal hypoxia&#8211;ischemia.&#160;(A) Representative power spectrogram from the ischemic parietal cortex electrode through the experimental timeline. (Amplitude color heat map scale x10&#8211;6). Arrows indicate the time that raw electroencephalogram tracings below the spectrogram represent. (B) Seizure behaviors for the entire experiment, postischemia/prehypoxia, during hypoxia, and posthypoxia. (C) Behavioral seizure score (BSS) and timing for all seizure events (n = 30 mice, each mouse has a unique symbol, each point is a discrete seizure event). 100% of mice seized during hypoxia (blue box; time = &#8722;60 minutes is the completion of carotid ligation, time=0 is the start of hypoxia). Thirteen percent died during hypoxia following a convulsive seizure (grade 5&#8211;6). This figure has been modified from Burnsed et al13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61346/61346fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61346/61346fig2v2.jpg" /> 
Figure 2: Characteristic electroencephalography (EEG) patterns during hypoxia ischemia.&#160;(A) EEG background from left to right: preinjury baseline, burst suppression during hypoxia, posthypoxia suppression. Recording from ipsilateral parietal cortex depth electrode. (B) Evolution of a seizure during hypoxia. Recording from ipsilateral hippocampal depth electrode. Shaded boxes (I-V) corresponded to expanded EEG excerpts on the right of (B). This figure has been modified from Burnsed et al13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61346/61346fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="bigfig" src="/files/ftp_upload/61346/61346fig03.jpg" /> 
Figure 3: Asymmetries in EEG background between ischemic and contralateral hemispheres.&#160;(A) Asymmetric power spectrogram in HI mice during hypoxia (45-minute period) in ischemic cortex (left) and contralateral cortex (right; amplitude scale x10&#8211;6). Burst suppression pattern and seizures in ischemic hemisphere, suppression in CL hemisphere. (B) Background suppression during hypoxia and reoxygenation in IL and CL hemispheres. All measurements of mean voltage taken from 10-second random excerpts of the encephalogram over the experimental time period (baseline, 30 minutes postligation, during hypoxia&#8212;15 minutes and 30 minutes after start, after reoxygenation&#8212;15 minutes and 60 minutes after start) were compared to baseline. Each animal&#8217;s baseline served as its own control, and data are reported as a percentage of baseline (n = 5 mice). Measurements were taken from cortical electrodes. This figure has been modified from Burnsed et al13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61346/61346fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

continuous-video-electroencephalogram-during-hypoxia-ischemia

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.3K Views.  University of Virginia. Dit manuscript beschrijft een methode voor continue video-EEG-opnames met behulp van meerdere diepte-elektroden bij neonatale muizen die hypoxie-ischemie ondergaan.

Video: Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to adequate numbers prior to transplantation. Identifying approaches to refine this process, to augment the production of all CNS cell types (i.e., neurons), and to possibly contribute to therapeutic cell therapy protocols is a high research priority. This report describes an easily applied in vivo &#8220;pre-conditioning&#8221; stimulus which can be delivered to awake, non-anesthetized animals. Thus, it is a non-invasive and non-stressful procedure. Specifically described are the procedures for exposing mouse or rat pups (aged postnatal day 1-8) to a brief (40-80 min) period of intermittent hypoxia (AIH). The procedures included in this video protocol include calibration of the whole-body plethysmography chamber in which pups are placed during AIH and the technical details of AIH exposure. The efficacy of this approach to elicit tissue-level changes in the awake animal is demonstrated through the enhancement of subsequent in vitro expansion and neuronal differentiation in cells harvested from the subventricular zone (SVZ). These results support the notion that tissue level changes across multiple systems could be observed following AIH, and support the continued optimization and establishment of AIH as a priming or conditioning modality for therapeutic cell populations.

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

This article describes the methodology for administering short periods of intermittent hypoxia to postnatal day 1-8 mouse or rat pups. This approach effectively elicits a robust tissue level &#8220;priming effect&#8221; on cultured neural progenitor cells that are harvested within 30 min of hypoxia exposure.

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to ...

Developmental Biology

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

Medicine

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

Genetics

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

Biology

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

Noninvasive Electrocardiography in the Perinatal Mouse

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both human and animal models of disease. Individual heart rate, rhythm, and regularity, combined with quantitative parameters collected from ECG, serve to assess the integrity of the cardiac conduction system as well as the integrated physiology of the cardiac cycle. This article provides a comprehensive description of the methods and techniques used to perform a noninvasive ECG on perinatal and neonatal mouse pups as early as the first postnatal day, without requiring the use of anesthetics. This protocol was designed to directly address a need for a standardized and repeatable method for obtaining ECG in newborn mice. From a translational perspective, this protocol proves to be entirely effective for characterization of congenital cardiopulmonary defects generated using transgenic mouse lines, and particularly for analysis of defects causing lethality at or during the first postnatal days. This protocol also aims to directly address a gap in the scientific literature to characterize and provide normative data associated with maturation of the early postnatal cardiac conduction system. This method is not limited to a specific postnatal timepoint, but rather allows for ECG data collection in neonatal mouse pups from birth to postnatal day 10 (P10), a window that is of critical importance for modeling human diseases in vivo, with particular emphasis on congenital heart disease (CHD).

Here, we present a noninvasive electrocardiography (ECG) protocol, optimized for early postnatal mice, that does not require the use of anesthetics.

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both ...

Echocardiographic Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular disease and is used to assess the impact of potential treatments in preclinical studies. Echocardiographic studies are usually conducted in young adult mice (i.e., 4-6 weeks of age). The evaluation of early neonatal cardiovascular function is not usually performed because of the small size of the mouse pups and the associated technical difficulties. One of the most important challenges is that the short length of the pups&#39; limbs prevents them from reaching the electrodes in the echocardiography platform. Body temperature is the other challenge, as pups are very susceptible to changes in temperature. Therefore, it is important to establish a practical guide for performing echocardiographic studies in small mouse pups to help researchers detect early pathological changes and study the progression of cardiovascular disease over time. The current work describes a protocol for performing echocardiography in mouse pups at the early age of 7 days old. The echocardiographic characterization of cardiac morphology, function, and coronary flow in neonatal mice is also described.

The present protocol describes the echocardiographic assessment of left ventricular morphology, function, and coronary blood flow in 7-day old neonate mice.

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular ...

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (&gt;12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term ...

Establishing a Mouse Model of Neonatal Hypoxic-Ischemic Brain Injury

Source: Kim, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55838">Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury.</a> J. Vis. Exp. (2017).
The video demonstrates the procedure for creating a mouse model to study neonatal hypoxic-ischemic brain injury. It outlines the steps for surgically inducing ischemia and subjecting the animal to hypoxia to exacerbate brain damage. Finally, it shows how to assess the resulting brain lesions, ensuring the mouse model is prepared for further scientific investigation.

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

Samenvatting

Meer video's verkennen