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 border="0" cellpadding="0" cellspacing="0" width="942">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">disposable subdermal needle electrodes</td>
			<td style="width:328px;">Technomed</td>
			<td style="width:119px;">TE/S43-438</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Genuine Grass Gold Disk Electrodes</td>
			<td>Natus</td>
			<td>FE5GH-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">neonatal hydrogel sensors</td>
			<td style="width:328px;">Natus</td>
			<td style="width:119px;">CZA00037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">positioning strips</td>
			<td style="width:328px;">Natus</td>
			<td style="width:119px;">OBM00047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">skin markers</td>
			<td style="width:328px;">Natus</td>
			<td style="width:119px;">CZN00011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Nu Prep skin prepping gel</td>
			<td>Weaver and Company</td>
			<td>10-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">contact gel Ten 20</td>
			<td>Weaver and Company</td>
			<td>10-20-4T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">BrainZ BRM3 Monitor</td>
			<td style="width:328px;">Natus</td>
			<td style="width:119px;"></td>
			<td>no longer available. New Monitor: CFM Olympic BrainZ Monitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">sensor adapter set</td>
			<td>Natus</td>
			<td>CZA00012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">skin disinfectant</td>
			<td style="width:328px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">swab</td>
			<td style="width:328px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">tape</td>
			<td style="width:328px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">cotton swab</td>
			<td style="width:328px;"></td>
			<td style="width:119px;"></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 ...

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38.2K 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

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 ...

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 ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Transcutaneous Microcirculatory Imaging in Preterm Neonates

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest capillaries, arterioles and venules. The magnifying effect visualizes the flow pattern of erythrocytes through these vessels.
It's non-invasive character makes it suitable to apply in (preterm) neonates, even in cardiorespiratory unstable patients. In adults and children, MI is mainly performed sublingually, but this is not possible in preterm infants as these cannot cooperate and the size of the probe is problematic. In preterm infants, MI is therefore performed transcutaneously. Their thin skin makes it possible to obtain high quality images of peripheral microcirculation. 
In this manuscript we will demonstrate the method of transcutaneous MI in preterm infants. We will focus on the different techniques and provide tips to optimize image quality. The highlights of software settings, safety and offline analysis are also addressed.

Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest ...

How to Administer Near-Infrared Spectroscopy in Critically ill Neonates, Infants, and Children

Near infrared spectroscopy (NIRS) calculates regional tissue oxygenation (rSO2) using the different absorption spectra of oxygenated and deoxygenated hemoglobin molecules. A probe placed on the skin emits light that is absorbed, scattered, and reflected by the underlying tissue. Detectors in the probe sense the amount of reflected light: this reflects the organ-specific ratio of oxygen supply and consumption - independent of pulsatile flow. Modern devices enable the simultaneous monitoring at different body sites. A rise or dip in the rSO2 curve visualizes changes in oxygen supply or demand before vital signs indicate them. The evolution of rSO2 values in relation to the starting point is more important for interpretation than are absolute values.
A routine clinical application of NIRS is the surveillance of somatic and cerebral oxygenation during and after cardiac surgery. It is also administered in preterm infants at risk for necrotizing enterocolitis, newborns with hypoxic ischemic encephalopathy and a potential risk of impaired tissue oxygenation. In the future, NIRS could be increasingly used in multimodal neuromonitoring, or applied to monitor patients with other conditions (e.g., after resuscitation or traumatic brain injury).

This protocol is designed to assist clinicians to measure regional tissue oxygenation at different body sites in infants and children. It can be used in situations where tissue oxygenation is potentially compromised, particularly during cardiopulmonary bypass, when using non-pulsatile cardiac-assist devices, and in critically ill neonates, infants and children.

Near infrared spectroscopy (NIRS) calculates regional tissue oxygenation (rSO2) using the different absorption spectra of oxygenated and deoxygenated ...

Evaluation of Capnography Sampling Line Compatibility and Accuracy when Used with a Portable Capnography Monitor

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment of end-tidal CO2 (ETCO2), its accuracy is commonly validated using commercial kits composed of a capnography monitor and its matching disposable nasal cannula sampling lines. The purpose of this study was to assess the compatibility and accuracy of cross-paired capnography sampling lines with a single portable bedside capnography monitor. A series of 4 bench tests were performed to evaluate the tensile strength, rise time, ETCO2 accuracy as a function of respiratory rate, and ETCO2 accuracy in the presence of supplemental O2. Each bench test was performed using specialized, validated equipment to allow for a full evaluation of sampling line performance. The 4 bench tests successfully differentiated between sampling lines from different commercial sources and suggested that due to increased rise time and decreased ETCO2 accuracy, not all nasal cannula sampling lines provide reliable clinical data when cross-paired with a commercial capnography monitor. Care should be taken to ensure that any cross-pairing of capnography monitors and disposable sampling lines is fully validated for use across respiratory rates and supplemental O2 flow rates commonly encountered in clinical settings.

The goal of this study was to evaluate the accuracy of capnography sampling lines used in conjunction with a portable bedside capnography monitor. Sampling lines from 7 manufacturers were evaluated for tensile strength, rise time, and ETCO2 accuracy as a function of respiratory rate or supplemental oxygen flow rate.

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

In Vitro Application of a Wireless Sensor in Flexion-Extension Gap Balance of Unicompartmental Knee Arthroplasty

Unicompartmental knee arthroplasty (UKA) is an effective treatment for end-stage anteromedial osteoarthritis (AMOA). The key to UKA is the flexion-extension gap balance, which is closely related to postoperative complications such as bearing dislocation, bearing wear, and arthritis progression. The traditional gap balance assessment is performed by indirectly sensing the tension of the medial collateral ligament by a gap gauge. It relies on the surgeon's feel and experience, which is imprecise and difficult for beginners. To accurately assess the flexion-extension gap balance of UKA, we developed a wireless sensor combination consisting of a metal base, a pressure sensor, and a cushion block. After osteotomy, the insertion of a wireless sensor combination allows the real-time measurement of intra-articular pressure. It accurately quantifies the flexion-extension gap balance parameters to guide further femur grinding and tibia osteotomy, to improve the accuracy of gap balance. We conducted an in vitro experiment with the wireless sensor combination. the results showed that there was a difference of 11.3 N after applying the traditional method of flexion-extension gap balance performed by an experienced expert.

In Vitro Application of a Wireless Sensor in Flexion-Extension Gap Balance of Unicompartmental Knee Arthroplasty

This protocol presents a cadaveric study of a wireless sensor used in medial unicompartmental knee arthroplasty. The protocol includes the installation of an angle measuring device, standardized Oxford unicompartmental knee arthroplasty osteotomy, preliminary assessment of flexion-extension balance, and application of the sensor to measure flexion-extension gap pressure.