We thank our nurses for their support and contributions to the making of the video.

aEEGs are conducted as part of the clinical routine in our hospital. The presented protocol follows the guidelines of the institution&#39;s human research ethics committee. Written informed consent regarding the filming and the publication of the material was collected from both parents of all infants that appear in the video.
1. Gather the Needed Supplies
<ol>
	<li>Connect the eEEG device to electric power in the place where the monitoring will take place and plug the module box to the aEEG device. &#160;&#160;</li>
	<li>Ensure that there are four electrodes for a two-channel aEEG and two electrodes for a single-channel aEEG. Choose either needle electrodes, gold cups, or hydrogel electrodes. Additionally, have one hydrogel electrode ready to serve as a reference electrode. 
	NOTE: Gold cups can be disinfected and reused for up to two years. Needle and hydrogel electrodes are single-use only. Needle electrodes can be used in infants at 23 weeks of gestation without causing skin lesions or infections. Here, the best results were achieved using needle electrodes in older infants as well.</li>
	<li>Prepare the following supplies: a positioning strip provided by the manufacturer (to help place the electrodes correctly), tape appropriate for use in neonates (e.g., viscose mull), skin disinfectant appropriate for use in neonates (e.g., alcohol-based or octenidinhydrocholoride-based), swabs, skin preparation gel, a module box, and contact gel for gold cups. 
	NOTE: Once disconnected from electrical power, the device will shut down, and the recording will need to be restarted. Some devices have internal batteries, however, and can be moved after being switched on or during the recording.</li>
</ol>
2. Apply the Electrodes
<ol>
	<li>Respecting the principals of minimal handling42,43,44, apply the electrodes during routine care or delivery room care. Wear (unsterile) gloves, a gown, a hood, and a mask, according to the institution&#39;s guidelines and the patient&#39;s infectious status.</li>
	<li>Prepare the skin for the reference electrode, as follows:
	<ol>
		<li>Disinfect the skin. Place skin preparation gel on a cotton swab until it is moist. Apply a few gentle strokes with the cotton swab, using very little pressure. Be very cautious in extremely premature infants between 23 and 25 weeks of gestation to avoid causing lesions on the immature skin.</li>
		<li>Place the reference electrode on the back or the chest of the infant.</li>
		<li>Place the measuring device on the infant&#39;s head, and line up the same letters/signs on the infant&#39;s tragus and sagittal suture; the two arrows indicate where to place the electrodes (positions C3, P3, C4, and P4 of the 10 - 20 system).</li>
	</ol>
	</li>
	<li>Place the electrodes on the infant&#39;s head, following the instructions, below, corresponding to the type of electrodes selected.</li>
	<li>Needle electrodes.
	<ol>
		<li>Disinfect the area indicated by the measuring device.</li>
		<li>Stretch the skin slightly and insert the needle tangentially, just underneath the skin at the markings, with the tip of the needle pointing in the caudal direction. Use tape to keep the electrode in place.</li>
		<li>Repeat the procedure for both/all four electrodes. 
		NOTE: Use needle electrodes in very premature infants, as rubbing for skin preparation is not necessary.</li>
	</ol>
	</li>
	<li>Gold cups.
	<ol>
		<li>Disinfect the area indicated by the measuring device.</li>
		<li>Prepare the skin in the marked areas, as described in step 2.2.</li>
		<li>Fill each cup with contact gel. Place the cup in the proper position, with the cable running towards the head end; tape it into place.</li>
	</ol>
	</li>
	<li>Hydrogel electrodes.
	<ol>
		<li>Disinfect the area indicated by the measuring device.</li>
		<li>Prepare the skin in the marked areas, as described in step 2.2.</li>
		<li>Place the electrodes with the cable running towards the head end. Fix the electrodes with tape in case they do not stay in place.</li>
	</ol>
	</li>
</ol>
3. Connect the Wires to the Monitor
<ol>
	<li>Insert the cables into the module box, as indicated by the legend on the box.</li>
	<li>The default starting screen includes impedance monitoring for all electrodes.</li>
	<li>Make sure that all electrodes are in place and that there is no mechanical contact between electrodes. If the impedance of one or more electrodes is not satisfactory, remove the corresponding electrode and perform one or two more strokes with the cotton swab, but do not apply more pressure.</li>
	<li>Start the recording when everything is set. 
	NOTE: Obligatory recording parameters are raw EEG and impedance. According to the device and clinical indication, additional options are burst suppression ratio, sharp transient intensity, and spectral edge frequency, among others. Standard parameters for review include raw EEG, amplitude-integrated EEG curve, and impedance. Depending on the device, there is the opportunity to view more features, like spectral edge frequency, or different forms of the presentation of the lower, mean, and upper border. Additional features are seizure detection and burst rate analysis.</li>
</ol>
4. Optional: Place a CPAP Hat
<ol>
	<li>If required, place a CPAP hat or head band on top of the aEEG electrodes.</li>
</ol>
5. Aspects to Keep in Mind During the Recording
<ol>
	<li>Regularly check for impedance and the dislocation of electrodes to obtain quality recordings. Also, check the infant for skin irritation to avoid lesions or infections.</li>
	<li>Mark events (e.g., handling, kangaroo care (skin-to-skin care), apnea with bradycardia, intubation, and administration of sedatives or opioids) to facilitate the identification of artifacts using the provided button on the screen of the cerebral function monitor. Leave aEEG electrodes in place during kangaroo care to resume the recording afterwards.</li>
	<li>Leave aEEG electrodes in place for intubation or other invasive measures to resume the recording later. In case the cables are not long enough to move the infant within the incubator, disconnect them from the module box and reconnect them after the procedure.</li>
</ol>
6. Review of the aEEG Tracing and Storing
<ol>
	<li>Review the tracing at the end of the recoding on the monitor or transfer it to an external storage device.</li>
</ol>

The authors have nothing to disclose.

The cerebral function monitor is an easily accessible and increasingly common device used to record amplitude-integrated EEGs in NICUs13. In routine care, the application of an aEEG takes 3 - 5 min.
Critical steps within this protocol are the correct placement of the electrodes on the head and the connection of the cables to the corresponding plug of the module box. Electrode placement needs to be preceded by thorough skin disinfection and preparation, especially for the reference electrode. In our experience, the best-quality recordings are achieved when the reference electrode is placed on the back of the infant. For troubleshooting, electrodes should be checked for dislocation in case of high impedance. If electrode dislocation has been disclosed and repetition of skin preparation fails, the electrode may need to be replaced. In case of an offset of the upper border, the reference electrode needs to be optimized. A high frequency and high amplitude of both the raw EEG and amplitude-integrated EEG are caused by muscle activity or interferences (e.g., high-frequency oscillation ventilation). This part of the tracing cannot be used for interpretation. If a flat trace occurs in a cerebrally healthy infant, the cross-cerebral tracing should be viewed. If this is normal, it is likely that liquids like sweat or ultrasound gel have caused bridging between two electrodes. In case of persisting problems, manufacturers have contact persons that will help to determine a solution and will even come to the NICU to check for underlying causes. In our experience, needle electrodes are the recommended type of electrodes in very premature infants. After thorough disinfection and gentle skin preparation of the reference electrode, we did not observe a significant number of infections, serious skin lesions, or bleeding events since the beginning of the large-scale use of this technique in our center in 2008 (an average of 60 - 80 very low birth weight infants per year, 1 - 5 recordings per infant). Since 2014, we have only used needle electrodes in all neonates, as we achieve the best results using this type of electrode.
Modifications of the aEEG are not commonly performed, but electrodes could be placed in any position on the head to acquire a desired tracing (from the international 10 - 20 system). In some cases, the electrode position may need to be adjusted (e.g., due to skin lacerations after vacuum extraction or cephalohematoma)45. For classification according to amplitudes, it is important to maintain a standard interelectrode distance, as a reduction in interelectrode distance results in a reduction of amplitude39,45. In case of extreme head sizes, such extremely premature infants (i.e., 23 - 24 weeks of gestation) or term infants with augmented head circumference due to hydrocephalus, the importance of the interelectrode distance for interpretation should be kept in mind. Another modification of the traditional aEEG is the continuously monitored limited-channel EEG18,46,47. The raw EEG curve derived from the cerebral function monitor can be assessed like a conventional EEG curve. In our center, we use this approach to answer special problems regarding newborn neuropediatric patients, in close collaboration with our pediatric neurologists.
The main limitation of the aEEG is the fact that only a small area of the brain surface is covered by the tracing. Thus, alterations of electrocortical activity in different areas of the brain surface may remain unnoticed13. Due to the time compression, short-lasting changes of cerebral activity are difficult to detect without using the raw EEG curve. Further interpretation of the raw EEG curve requires knowledge about the conventional EEG or close cooperation with neurophysiologists or pediatric neurologists. Last, but not least, there are several external and internal factors that cause alterations in the aEEG band that must be kept in mind when interpreting the tracing.
Nonetheless, the aEEG offers the possibility for continuous cerebral function monitoring in neonates. It is easily accessible, and interpretation is not difficult. As it contains less information than a conventional EEG, it cannot replace this technique. Rather, it complements the existing means for cerebral diagnostics, such as EEG, ultrasound, and magnetic resonance imaging. There is good evidence for the prediction of outcome after birth asphyxia in term infants, and the aEEG has been established as a tool to identify infants who will benefit from cooling8. In preterm infants, there is also good evidence that long-term neurological outcome can be predicted by early aEEG recordings15,16,17,18,19,20. However, to date, this knowledge does not result in consequences for clinical decision making in this population of infants. For the future, it is likely that cerebral function monitoring will become a standard tool in NICUs, as well as in secondary centers and pediatric intensive care units.

aEEG was originally developed as a bedside monitor for adult intensive care1. The first publications detailing its use in neonates date back to the late 1980s2,3. In early years, its clinical use was mainly for the detection of cerebral seizure activity, surveillance of antiepileptic drug treatment4, and prediction of cerebral outcome after birth asphyxia5,6,7,8,9. Infants with birth asphyxia who did not show severe suppression of background activity and seizure activity had a more favorable outcome if they were cooled8, but research on this topic is still ongoing10,11,12. Over the past 30 years, cerebral function monitoring in neonates has become more widespread in NICUs13. Nowadays, it is increasingly being used in the preterm infant population. aEEG has been proven to be a safe method for cerebral function monitoring, even in extremely preterm infants, and is generally well-accepted by NICU staff14. Several studies showed a correlation between early aEEG recordings and neurodevelopmental outcomes in preterm infants15,16,17,18,19,20.
aEEG is based upon conventional electroencephalography that is recorded with two or four scalp electrodes, depicting the amplitude of the raw EEG on a time-compressed semi-logarithmic scale1. The signal from two or four electrodes placed in the C3, P3, C4, and P4 positions of the international 10-20 system is passed through a bandpass filter, which enhances frequencies between 2 and 15 Hz. Frequencies under 2 Hz and above 15 Hz are attenuated in order to eliminate artefacts, such as sweating, movement, muscle activity, and electrical interference, as much as possible1,4. Further processing includes filtering, rectification, smoothing, semi-logarithmic amplitude compression, and time compression. Amplitudes &#60;10 &#181;V are displayed on a linear scale and amplitudes &#62;10 &#181;V on a logarithmic scale21. The lowest-detected amplitude is shown as the lower border, and the highest amplitude is shown as the upper border21. By this means, even small changes in the lower amplitude remain visible, while an overloading of the display at high amplitudes is avoided21 (Figure 1). Due to time compression, 5 - 6 cm on the time scale represents 1 h, thus making the review of brain activity for hours and even days possible1,4,13.
The visible information in the aEEG tracing is limited to changes of the amplitude. Modern devices offer the possibility of viewing the raw EEG, so the frequency and morphology of the raw EEG curve can also be considered for interpretation. This helps to distinguish between artefacts and real seizure activity during suspicious sections of the aEEG band4. Some aEEG devices can record a simultaneous video of the patient for even better identification of seizures and artifacts. Electrode impedance is monitored during the entire recording21. In two-channel aEEG devices that use four electrodes, the investigator can switch between two intraparietal curves or one transcerebral curve (Figure 2). Depending upon the manufacturer, software offers additional features, like seizure detection, burst rate analysis, electromyography, etc. It is also possible to derive an aEEG from a full-channel EEG device that offers video recording, electromyography, electrooculography, electrocardiography, etc.
The reduction of the electrophysiological information and time compression makes continuous monitoring and bedside interpretation possible without requiring specific knowledge about EEG. Because of the long recording time, even subclinical seizure activity can be detected, which would otherwise remain unnoticed4,22 because conventional EEG monitoring for very long time periods is not available to date in NICUs. It must be considered, though, that not all pathological alterations, like seizures, are found due to the small area of brain surface covered by the recording13. Therefore, aEEG is not meant to replace conventional EEG, but to complement it13.
Electrocortical activity, as reflected by the aEEG background pattern, changes according to the infant&#39;s gestational age4,23,24,25. In term infants and late preterm infants, the background pattern is mainly continuous with a lower amplitude above 5 &#181;V4. During quiet sleep, the background pattern becomes more discontinuous26. In very preterm infants, the dominating background pattern is discontinuous: episodes of high activity (i.e., high-amplitude bursts) alternate with episodes of low-amplitude activity27. This physiological pattern must be distinguished from a burst suppression pattern, which is pathological27. With increasing gestational age, aEEG and background patterns become more continuous, and the duration of continuous activity increases27,28,29. Developing and existing pathological conditions can also be displayed by the aEEG tracing (e.g., the development of an intraventricular hemorrhage and periventricular leukomalacia is associated with acute disturbances in the background activity)30,31. Severe meningitis can cause a flat trace.
The qualitative interpretation of aEEG generally includes three categories: classification of the background pattern, sleep-wake cycling, and the presence of seizures. Several authors have made suggestions for classifications and scores that describe brain maturation16,21,24,25. The quantitative analysis of aEEG is less common, even though it is possible in modern devices, and few research groups made use of this approach32,33,34. We would like to briefly present 3 different approaches to the qualitative and semi-quantitative assessment of aEEG tracings:
Hellstr&#246;m-Westas:21
The assessment of the tracing is solely qualitative, and the results are not transformed into a score. The classification allows for the description of pathological conditions. Normative values for gestational ages have been published to help interpret whether a pattern is adequate for the age21: (1) background patterns: continuous normal voltage (physiological), discontinuous normal voltage (physiological in preterm infants), burst suppression pattern (pathological), continuous low voltage (pathological), and flat trace (pathological); (2) sleep-wake cycling: none, imminent, mature (physiological/pathological, depending on the infant&#39;s age); and (3) seizure activity: none, single seizures, repetitive seizures, and status epilepticus.
Burdjalov:25
The approach of this classification is the qualitative assessment of the tracing and its transformation into a score. The score rises with gestational age, and normative score values for each corresponding gestational age have been published: (1) 0 - 2 points for continuity, (2) 0 - 5 points for sleep-wake cycling, (3) 0 - 2 points for the amplitude of the lower border, (4) 0 - 4 points for the bandwidth, and (5) 0 - 13 points for the total score.
Olischar/Klebermass:16,24
Percentiles regarding the percent duration of background patterns (i.e., discontinuous normal voltage, discontinuous low voltage, and continuous normal voltage) and burst rate were developed for gestational ages. Tracings are evaluated for an age-adequate background pattern, the presence of sleep-wake cycling, and the presence of seizure activity (i.e., repetitive seizures or status epilepticus). Then, the tracings are classified into a graded score as follows: (1) normal aEEG (all categories normal), (2) moderately abnormal (1 out of 3 categories classified as abnormal), and (3) severely abnormal (2 or 3 out of 3 categories classified as abnormal). This score has been shown to have a predictive value for neurodevelopmental outcome at 3 years of corrected age.
Changes in the aEEG tracing are caused by numerous extracortical factors, such as changes in cerebral blood flow, medication (e.g., opiates, sedatives, and caffeine), acidosis, changes in carbon dioxide tension, clinical conditions (e.g., hypogylcemia, sepsis, meningitis, and patent ductus arteriosus), etc.21,32,35,36,37,38. The aEEG band itself is rather insensitive to changes of impedance, but significant changes are observed in terms of electrode distance and localization39. Artifacts may pose a problem for interpretation: absolute values of the amplitude change as a consequence of scalp edema or interelectrode distance39,40. Interferences caused by ECG, high-frequency oscillation ventilation, muscle activity, infant movement, or handling may result in a lower border increase40. In modern devices, this can be partially avoided by the simultaneous recording of raw EEG and aEEG and by marking the beginning and end of handling. Liquids (e.g., sweat or ultrasound gel) can lead to connections between electrodes, feigning a flat trace pattern. Approximately 12% of the recording time in long-term aEEGs are altered due to artifacts, 55% caused by electrical interferences and 45% being movement artifacts41.

<table><tbody><tr><td>disposable subdermal needle electrodes</td><td>Technomed</td><td>TE/S43-438</td><td></td></tr><tr><td>Genuine Grass Gold Disk Electrodes</td><td>Natus</td><td>FE5GH-03</td><td></td></tr><tr><td>neonatal hydrogel sensors</td><td>Natus</td><td>CZA00037</td><td></td></tr><tr><td>positioning strips</td><td>Natus</td><td>OBM00047</td><td></td></tr><tr><td>skin markers</td><td>Natus</td><td>CZN00011</td><td></td></tr><tr><td>Nu Prep skin prepping gel</td><td>Weaver and Company</td><td>10-30</td><td></td></tr><tr><td>contact gel Ten 20</td><td>Weaver and Company</td><td>10-20-4T</td><td></td></tr><tr><td>skin disinfectant</td><td></td><td></td><td></td></tr><tr><td>tape</td><td></td><td></td><td></td></tr><tr><td>cotton swab</td><td></td><td></td><td></td></tr><tr><td>Braintrend&lt;sup&gt;&amp;#174;&lt;/sup&gt; aEEG Monitor</td><td>aEEG Monitor</td><td></td><td></td></tr></tbody></table>

application of an amplitude-integrated eeg monitor (cerebral function monitor) to neonates

Amplitude-integrated EEG (aEEG) is an easily accessible technique to monitor the electrocortical activity in preterm and term infants in neonatal intensive care units (NICUs). This method was first used to monitor newborns after asphyxia, providing information about future neurological outcomes. The aEEG is also helpful to select newborns who benefit from cooling. The aEEG monitoring of preterm infants is becoming more widespread, as various studies have shown that neurodevelopmental outcome is related to early aEEG tracings. Here, we demonstrate the application of the aEEG monitoring system and present typical patterns that depend upon gestational age and pathophysiological conditions. Furthermore, we mention pitfalls in the interpretation of the aEEG, as this method requires accurate fixation and localization of the electrodes. Additionally, the raw EEG can be used to detect neonatal seizures or to identify aEEG application problems. In conclusion, aEEG is a safe and generally well-tolerated method for the bedside monitoring of neonatal cerebral function; it can even provide information about long-term outcome.

Figure 2 shows a typical view of an aEEG monitor. Continuous and discontinuous normal voltage patterns are considered physiological background patterns in term and preterm infants, respectively (Figure 3 and Figure 4). A burst suppression pattern, continuous low-voltage pattern, and a flat trace are pathological background patterns (Figure 5, Figure 6, Figure 7).
Seizures in term infants have a characteristic shape, with a sudden rise of both the lower and upper border (Figure 8). In preterm infants, however, seizures can be camouflaged by the discontinuous pattern and may only be detected by viewing the raw EEG (Figure 9).
Liquid bridging can cause an apparent flat trace (Figure 10). Usually, this happens in the two-channel aEEG (intraparietal curves). If the cross-cerebral aEEG is physiological, while the intraparietal curve shows a flat trace, the electrodes should be checked for liquids. Electrical interference, movements, and handling can lead to an apparent seizure or even to an apparent status epilepticus. If this happens, impedance and the reference electrode should be checked, and the raw EEG should be viewed (Figure 11). Another reason for an elevation of both the lower and upper border is the displacement of the reference electrode.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55985/55985fig1.jpg" /> 
Figure 1. Formation of the aEEG Tracing. 
The signal from the raw EEG (upper curve) is processed, resulting in the amplitude-integrated EEG band (lower curve). High amplitudes form the upper border, whereas low amplitudes form the lower border. While strong variation in the height of the amplitude leads to a broad aEEG band, the aEEG band is narrow if there is little variation in the height of amplitude. The scale of the y-axis is linear up to 10 &#181;V and logarithmic above 10 &#181;V. <a href="http://cloudflare.jove.com/files/ftp_upload/55985/55985fig1large.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/55985/55985fig2.jpg" /> 
Figure 2. Typical Display of an aEEG Monitor. 
The upper half of the monitor displays the raw EEG curve (the displayed section equals 10 s). On the left display, the lower half shows the unilateral aEEG tracing (the displayed section equals approximately 3 h). On the right display, the corresponding cross-cerebral tracing is shown. The cursor indicates the section of the amplitude-integrated tracing from the raw EEG. <a href="http://cloudflare.jove.com/files/ftp_upload/55985/55985fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55985/55985fig3v2.jpg" /> 
Figure 3. Continuous Normal Voltage Pattern. 
Continuous background pattern with sleep-wake cycling. The x-axis equals time (one square = 10 min). <a href="http://cloudflare.jove.com/files/ftp_upload/55985/55985fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55985/55985fig4.jpg" /> 
Figure 4. Discontinuous Normal Voltage Pattern. 
Discontinuous background pattern with imminent sleep-wake cycling. The x-axis equals time (one square = 10 min). <a href="http://cloudflare.jove.com/files/ftp_upload/55985/55985fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55985/55985fig5.jpg" /> 
Figure 5. Burst Suppression Pattern. 
Burst suppression pattern, with the lower amplitude continuously low and without alteration. The x-axis equals time (one square = 10 min). 
From Bruns, N. Amplituden-integriertes EEG bei extrem unreifen Fr&#252;hgeborenen in den ersten 4 Lebenswochen.&#160;http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000036576 (2012). <a href="http://cloudflare.jove.com/files/ftp_upload/55985/55985fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55985/55985fig6.jpg" /> 
Figure 6. Flat Trace. 
Flat trace on both sides in a term infant with severe meningoencephalitis. The x-axis equals time (one square = 10 min). 
From Bruns, N. Amplituden-integriertes EEG bei extrem unreifen Fr&#252;hgeborenen in den ersten 4 Lebenswochen.&#160;http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000036576 (2012). <a href="http://cloudflare.jove.com/files/ftp_upload/55985/55985fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55985/55985fig7.jpg" /> 
Figure 7. Continuous Low-voltage Pattern. 
Continuous low-voltage pattern without sleep-wake cycling. The x-axis equals time (one square = 10 min). 
From Bruns, N. Amplituden-integriertes EEG bei extrem unreifen Fr&#252;hgeborenen in den ersten 4 Lebenswochen.&#160;http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000036576 (2012). <a href="http://cloudflare.jove.com/files/ftp_upload/55985/55985fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/55985/55985fig8.jpg" /> 
Figure 8. Seizures in Term Infants. 
Typical depiction of a seizure in the aEEG: a sudden rise of the lower and upper margin is followed by a short period of decreased activity. Repetitive seizures for approximately 3.5 h. The x-axis equals time (one square = 10 min). 
From Bruns, N. Amplituden-integriertes EEG bei extrem unreifen Fr&#252;hgeborenen in den ersten 4 Lebenswochen.&#160;http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000036576 (2012). <a href="http://cloudflare.jove.com/files/ftp_upload/55985/55985fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/55985/55985fig9.jpg" /> 
Figure 9. Seizures in Preterm Infants. 
Without the raw EEG, the hypersynchronous activity in both hemispheres would remain undetected. The x-axis equals time (one square = 10 min). 
From Bruns, N. Amplituden-integriertes EEG bei extrem unreifen Fr&#252;hgeborenen in den ersten 4 Lebenswochen.&#160;http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000036576 (2012). <a href="http://cloudflare.jove.com/files/ftp_upload/55985/55985fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/55985/55985fig10.jpg" /> 
Figure 10. Apparent Flat Trace. 
In the unilateral tracings, there appears to be a pathological flat trace pattern in an infant without cerebral injury. The cross-cerebral tracing shows a physiological discontinuous background pattern with short sections of continuous activity. In this case, the flat trace is an artifact caused by liquid bridging between electrodes (especially hydrogel electrodes). The x-axis equals time (one square = 10 min). 
From Bruns, N. Amplituden-integriertes EEG bei extrem unreifen Fr&#252;hgeborenen in den ersten 4 Lebenswochen.&#160;http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000036576 (2012). <a href="http://cloudflare.jove.com/files/ftp_upload/55985/55985fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/55985/55985fig11.jpg" /> 
Figure 11. Apparent Seizures. 
This image shows high-frequency activity over a long period of time. Without seeing the raw EEG curve, status epilepticus is indicated. This artifact is caused by muscle activity. The x-axis equals time (one square = 10 min). 
From Bruns, N. Amplituden-integriertes EEG bei extrem unreifen Fr&#252;hgeborenen in den ersten 4 Lebenswochen.&#160;http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000036576 (2012). <a href="http://cloudflare.jove.com/files/ftp_upload/55985/55985fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Application of an Amplitude-integrated EEG Monitor Cerebral Function Monitor to Neonates at JoVE.com

Application of an Amplitude-integrated EEG Monitor Cerebral Function Monitor to Neonates

Here, we show how to apply amplitude-integrated electroencephalography to monitor cerebral function in neonates.

Application of an Amplitude-integrated EEG Monitor (Cerebral Function Monitor) to Neonates

Amplitude-integrated EEG (aEEG) is an easily accessible technique to monitor the electrocortical activity in preterm and term infants in neonatal ...

application-an-amplitude-integrated-eeg-monitor-cerebral-function

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JoVE Journal

Medicine

38.3K Views.  University Duisburg-Essen. Here, we show how to apply amplitude-integrated electroencephalography to monitor cerebral function in neonates.

Video: Application of an Amplitude-integrated EEG Monitor Cerebral Function Monitor to Neonates

Infant Auditory Processing and Event-related Brain Oscillations

Rapid auditory processing and acoustic change detection abilities play a critical role in allowing human infants to efficiently process the fine spectral and temporal changes that are characteristic of human language. These abilities lay the foundation for effective language acquisition; allowing infants to hone in on the sounds of their native language. Invasive procedures in animals and scalp-recorded potentials from human adults suggest that simultaneous, rhythmic activity (oscillations) between and within brain regions are fundamental to sensory development; determining the resolution with which incoming stimuli are parsed. At this time, little is known about oscillatory dynamics in human infant development. However, animal neurophysiology and adult EEG data provide the basis for a strong hypothesis that rapid auditory processing in infants is mediated by oscillatory synchrony in discrete frequency bands. In order to investigate this, 128-channel, high-density EEG responses of 4-month old infants to frequency change in tone pairs, presented in two rate conditions (Rapid: 70 msec ISI and Control: 300 msec ISI) were examined. To determine the frequency band and magnitude of activity, auditory evoked response averages were first co-registered with age-appropriate brain templates. Next, the principal components of the response were identified and localized using a two-dipole model of brain activity. Single-trial analysis of oscillatory power showed a robust index of frequency change processing in bursts of Theta band (3 - 8 Hz) activity in both right and left auditory cortices, with left activation more prominent in the Rapid condition. These methods have produced data that are not only some of the first reported evoked oscillations analyses in infants, but are also, importantly, the product of a well-established method of recording and analyzing clean, meticulously collected, infant EEG and ERPs. In this article, we describe our method for infant EEG net application, recording, dynamic brain response analysis, and representative results.

High-density electroencephalography (dEEG) is being used increasingly to study brain development and plasticity in the early years of life. Here we present an application of sophisticated analysis techniques that builds on traditional EEG recording to understand the oscillatory dynamics of rapid auditory processing in the infant brain.

Rapid auditory processing and acoustic change detection abilities play a critical role in allowing human infants to efficiently process the fine spectral ...

Behavior

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

Neuroscience

Preterm EEG: A Multimodal Neurophysiological Protocol

Since its introduction in early 1950s, electroencephalography (EEG) has been widely used in the neonatal intensive care units (NICU) for assessment and monitoring of brain function in preterm and term babies. Most common indications are the diagnosis of epileptic seizures, assessment of brain maturity, and recovery from hypoxic-ischemic events. EEG recording techniques and the understanding of neonatal EEG signals have dramatically improved, but these advances have been slow to penetrate through the clinical traditions. The aim of this presentation is to bring theory and practice of advanced EEG recording available for neonatal units. 
In the theoretical part, we will present animations to illustrate how a preterm brain gives rise to spontaneous and evoked EEG activities, both of which are unique to this developmental phase, as well as crucial for a proper brain maturation. Recent animal work has shown that the structural brain development is clearly reflected in early EEG activity. Most important structures in this regard are the growing long range connections and the transient cortical structure, subplate. Sensory stimuli in a preterm baby will generate responses that are seen at a single trial level, and they have underpinnings in the subplate-cortex interaction. This brings neonatal EEG readily into a multimodal study, where EEG is not only recording cortical function, but it also tests subplate function via different sensory modalities. Finally, introduction of clinically suitable dense array EEG caps, as well as amplifiers capable of recording low frequencies, have disclosed multitude of brain activities that have as yet been overlooked.
In the practical part of this video, we show how a multimodal, dense array EEG study is performed in neonatal intensive care unit from a preterm baby in the incubator. The video demonstrates preparation of the baby and incubator, application of the EEG cap, and performance of the sensory stimulations.

This video explains the background theory of the neonatal EEG activity and the sensory responses, followed by a live demonstration of their recording in neonatal intensive care unit.

Since its introduction in early 1950s, electroencephalography (EEG) has been widely used in the neonatal intensive care units (NICU) for assessment and ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Electrophysiological Measurement of Noxious-evoked Brain Activity in Neonates Using a Flat-tip Probe Coupled to Electroencephalography

Pain is an unpleasant sensory and emotional experience. In non-verbal patients, it is very difficult to measure pain, even with pain assessment tools. Those tools are subjective or determine secondary physiological indicators which also have certain limitations particularly when exploring the effectiveness of analgesia. As cortical processing is essential for pain perception, brain activity measures may provide a useful approach to assess pain in infants. Here we present a method to assess nociception with electrophysiological brain activity recordings optimized for the use in newborn infants. To produce highly standardized and reproducible noxious stimuli we applied mechanical stimulation with a flat-tip probe, e.g., PinPrick, which is not skin-breaking and does not cause behavioral distress. The noxious-evoked potential allows the objective measurement of nociception in non-verbal patients. This method can be used in newborn infants as early as 34 weeks of gestational age. Moreover, it could be applied in different situations such as measuring the efficacy of analgesic or anesthetic drugs.

Measuring pain in non-verbal patients is a challenge. In this study we combine EEG recording with stimulation using a flat-tip probe to detect noxious-evoked brain activity in an objective manner.

Pain is an unpleasant sensory and emotional experience. In non-verbal patients, it is very difficult to measure pain, even with pain assessment tools. ...

How to Obtain Reliable Visual Event-related Potentials in Newborns

The present study discusses the characteristics of visual event-related potentials (VEPs) and outlines methodological steps for obtaining reliable measurements in newborns. Obtaining high-quality, reliable VEPs is crucial for the early detection of abnormal development of the central nervous system in at-risk newborns, and for implementing successful early interventions. Recommendations are based on a previous study which showed that when post-conceptional age, polysomnography-identified sleep stages, and light-emitting diodes (LEDs) googles as the luminous source are controlled, no more than 4 repetitions of VEP averages are required to obtain replicable recordings, variability decreases, and reliable VEPs can be obtained. By controlling for these sources of variability and using statistical analyses, we were able to clearly and reliably identify the amplitude and latency of three main components (NII, PII and NIII) present in 100% of newborns (n = 20) during active sleep. Recording VEPs during awake states, quiet sleep and transitional sleep is not recommended because VEP morphology may differ significantly from one average to the next, leading to the risk of misleading clinical prognoses. Moreover, it is easier to obtain VEPs during active sleep because this state can be clearly and reliably identified at this stage of development, sleep cycles are short enough to allow measurements to be taken in a reasonable time, and the method does not require new o expensive equipment.

Several important points for obtaining high-quality reliable visual evoked potentials (VEPs) in newborns while minimizing variability and the risk of misleading prognoses are presented.

The present study discusses the characteristics of visual event-related potentials (VEPs) and outlines methodological steps for obtaining reliable ...

Using a Portable Amplitude-Integrated Electroencephalogram for Infant Transport

Amplitude-Integrated EEG in Infants at Risk of Hypoxic-Ischemic Encephalopathy: A Feasibility Study in Road and Air Transport in Western Australia

Infants at risk of HIE require early identification and initiation of therapeutic hypothermia (TH). Earlier treatment with TH is associated with better outcomes. aEEG is frequently used when making the decision whether to commence TH. As this is often limited to tertiary centers, TH may be delayed if the infant requires transport to a center that provides it. We aimed to provide a method for the application of amplitude-integrated electroencephalogram (aEEG) and to determine the feasibility of acquiring clinically meaningful information during transport. All infants &#8805;35 weeks, at risk of HIE at referral, were eligible for inclusion. Scalp electrodes were placed in the C3-C4; P3-P4 position on the infant&#39;s scalp and connected to the aEEG amplifier. The aEEG amplifier was, in turn, connected to a clinical tablet computer with EEG software to collect and analyze aEEG information. Recordings were reviewed by the chief principal investigator and two independent reviewers (blinded) for background trace and artifact. Predefined criteria for data quality were set to movement artifacts and software impedance notifications. Surveys were completed by healthcare staff and parents for acceptability and ease of use.

Author Spotlight: Assessing the Feasibility of Using Amplitude-Integrated EEG During Neonatal Transport

Here, we present a feasibility study to assess a portable amplitude-integrated electroencephalogram (aEEG) recording system during the transport of infants with suspected hypoxic ischemic encephalopathy (HIE).

Infants at risk of HIE require early identification and initiation of therapeutic hypothermia (TH). Earlier treatment with TH is associated with better ...

Measuring Pain-Evoked Brain Activity in Neonates Using a Flat-Tip Probe Coupled to EEG

Source: Klarer, N. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/56531">Electrophysiological Measurement of Noxious-evoked Brain Activity in Neonates Using a Flat-tip Probe Coupled to Electroencephalography.</a>&#160;J. Vis. Exp. (2017).
This video demonstrates a method for measuring pain-evoked brain activity in infants. It outlines the procedure for positioning electrodes on the infant&#39;s head and applying pain stimuli to the infant&#39;s hand using a flat-tip probe. The video also shows the EEG recording process to identify brain activity in response to the stimuli.

Pomiar aktywności mózgu wywołanej bólem u noworodków za pomocą sondy z płaską końcówką sprzężoną z EEG

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...