Name: interictal high frequency oscillations detected with simultaneous magnetoencephalography and electroencephalography as biomarker of pediatric epilepsy
Uploaded: 2016-12-06T15:00:00
Duration: 10 min 22 s
Description: Watch this Scientific Journal Video about Interictal High Frequency Oscillations Detected with Simultaneous Magnetoencephalography and Electroencephalography as Biomarker of Pediatric Epilepsy at JoVE

This work is supported by the Research Grants Program of the Epilepsy Foundation &amp; American Epilepsy Society and the Faculty Career Development Fellowship of Harvard Medical School, Office for Faculty Development.

The authors have nothing to disclose.

Converging evidence from animal and human studies has shown that HFOs are a new potential biomarker for epileptogenic tissue. Despite this evidence, HFOs have very limited use in clinical practice for the diagnosis or monitoring of epilepsy, mostly because: (i) there is no formal and global definition for HFOs; (ii) different research groups use different methodology for recording and analyzing the data; (iii) the noninvasive detection of HFOs with neuroimaging techniques is challenging; and (iv) the review process of HFOs is time-consuming and impractical, especially for multichannel EEG or MEG recordings with a high number of sensors. In an effort to provide a global standardized methodology that promotes the systematic use of HFOs in clinical practice, the methodology that is followed at Boston Children&#39;s Hospital for the noninvasive recording, detection, and localization of interictal HFOs from pediatric patients with epilepsy is presented. Representative results of HFOs detected with simultaneous scalp EEG and MEG from two children with medically refractory epilepsy are also presented.
Critical steps within the protocol
The proposed methodology includes the following critical steps: (i) the performance of high Signal-to-Noise-Ratio (SNR) EEG and MEG simultaneous recordings of interictal activity from pediatric patients with medically refractory epilepsy (steps 2.1.1 and 2.1.2); (ii) the careful preprocessing and selection of data with interictal discharges (steps 3.1 and 3.2); (iii) the visual review of the identified HFOs events with high specificity (steps 4.3.1, 4.3.2, and 4.3.3); and (iv) the reliable localization of the HFOs using an appropriate localization method (step 5.2).
The most critical step in this protocol is the visual review of the HFO events identified by the automatic detector. A rigorous review of the automatically detected HFOs is crucial to discard HFOs of noncerebral origin. However, fatigue or distraction of the human reviewer during the visual inspection of multichannel EEG and MEG data may lead to errors, reducing the specificity of the detection process.
Modifications and troubleshooting
We avoid the use of the Signal Space Projection (SSP) and Signal Space Separation (SSS) methods72,73 in order to ensure that there was no distortion of the HFO activity from their application. These methods are often used by most of the users of the particular MEG vendor to suppress external interferences and to correct for head movements72. Further studies are necessary in order to ensure that the application of these methods do not affect or distort the HFO activity or do not produce spurious effects that may resemble human HFOs. Minor modifications of the minimum threshold of the z-score of the signal envelope (step 4.1.1.3) and the threshold of activation values (step 5.2.6) may be needed to improve the sensitivity of the algorithm in the detection of HFOs and restrict the localization of the HFOs zone in a more focal area.
Limitations of the technique
The described method presents limitations that should be further addressed in future studies. First, it does not consider HFOs occurring only in the MEG or EEG signals, and it does not include the automatic detection of HFOs in the MEG signals, which implies that some actual low SNR MEG HFOs might escape visual inspection74. Furthermore, the sensitivity and specificity of the proposed method to detect the HFOs and its ability to localize them with high accuracy should be validated with simultaneous recordings of scalp EEG, MEG, and iEEG75. Our data have shown that single ECDs indicated an extended irritative zone compared to the focal HFOs zone. However, when the ECDs were averaged, then the dipole location was quite close to the HFO zone for both patients. Our data are indicative of the specificity of the 2 methods showing a possible higher specificity of the HFO zone for epileptogenicity (particularly for patient 2 for whom the HFO zone was overlapping with the SOZ) compared to the irritative zone, although secure conclusions cannot be drawn from such a small cohort of patients. More importantly, the localization of the HFO sources does not directly imply localizing the EZ that is responsible for seizures. Our findings should be validated against the outcome of the epilepsy surgery that we plan to do in a future study. Finally, to record the EEG data, a 70-channel system was used. Yet, in most centers the standard clinical EEG setting is used that records data from 19 electrodes placed according to the 10 - 20 system. More advanced pediatric EEG systems with much higher number of channels (up to 256) are currently available in the market. The use of these systems may further improve the localization accuracy of the HFOs zone detected with scalp EEG.
Significance of the technique with respect to existing/alternative methods
To our best knowledge, this is the first study that reports the noninvasive localization of interictal HFOs with simultaneous EEG and MEG, and also investigates the concordance of the localization results with those from intracranial recordings. The noninvasive recording, detection, and localization of HFOs is challenging. This is because HFOs are very weak signals generated by small brain regions on the order of cubic millimeters16,76 and furthermore hindered by noise and brain background activity. A recent study proposed that HFOs recorded non-invasively with scalp EEG represent the sum of activity of multiple spatially distributed focal and coherent sources60. So far, few studies28,29,37,38,60 managed to show that HFOs can be detected non-invasively using scalp EEG and MEG; even fewer localized this activity by solving the inverse problem37-38.
Here, evidence of interictal HFOs are presented which have been detected with simultaneous scalp EEG and MEG from two pediatric patients with epilepsy. HFOs were localized by using a previously described framework38. The representative data suggest that the noninvasive localization of interictal HFOs is feasible by using source imaging techniques performed on either scalp EEG or MEG recordings, assuming that an appropriate localization technique is used. This is in line with a previous study that used a phantom construction resembling HFOs generators, which indicates that HFOs can be noninvasively detected and accurately localized with MEG32.
The detection and labeling of interictal HFOs is traditionally performed through the visual inspection of data from human EEG experts. Although this approach is often regarded as the gold standard, it presents serious limitations since it has poor inter-reviewer reliability77,78, and is not applicable to large MEG and EEG datasets with high number of sensors. Crucial to the application of HFOs in clinical practice is the development of algorithms that detect the HFOs automatically from scalp recordings reducing the need for human input. The visual identification of scalp HFOs is in fact quite challenging due to: (i) the low SNR of HFOs on the scalp; (ii) the lower rates of HFOs in scalp recordings compared to intracranial ones, which implies the analysis of much longer recording times; and (iii) the high number of channels to analyze, particularly in high-density EEG or MEG. Several algorithms for automatic and semi-automatic detection of HFOs have been proposed in the last decade54. Earlier detectors relied on thresholds in the time domain, in order to identify events that can be distinguished from ongoing background activity49,80. Recent advances suggest also incorporating information from the frequency domain, assuming that an HFO must appear as a short-lived event with an isolated spectral peak at a distinct frequency50,56,81. Semi-automated methods seem to be the most appropriate approach for the application of HFOs in clinical practice. These methods involve 2 stages: (i) initial automatic detection of events that has high sensitivity, and (ii) visual review of events by an expert, which has high specificity. This approach provides higher specificity compared to the fully automated methods and ensures that the final reviewed events are actual HFOs of cerebral origin.
Here, a semi-automated method is presented that enables the detection of HFOs from interictal scalp EEG and MEG recordings. The proposed method extends previously described techniques for the detection of HFOs from scalp EEG60 by incorporating in the identification criteria two important features: (i) the automatic time-frequency analysis of the HFO events; and (ii) the temporal concurrence of HFOs events in both MEG and EEG recordings.
Future applications or directions after mastering this technique
The reliable localization of HFOs with non-invasive neuroimaging methods, such as scalp EEG and MEG, is critical. Mastering, improving, and validating the proposed protocol will provide physicians with a reliable, noninvasively recordable biomarker for the identification of the EZ. The development of such a biomarker has the potential to reduce the requirement for long-term monitoring and invasive intracranial recordings leading to a significant improvement in the presurgical evaluation procedure in pediatric patients. It would not only help to identify the epileptogenic tissue for surgery, but would also permit definitive differential diagnosis of epilepsy from acute symptomatic seizures, requiring an entirely different treatment approach, and from non-epileptic seizures sparing the need for long-term monitoring in some patients. Further, this might allow assessment of efficacy of therapeutic interventions without waiting for another seizure to occur.

Pediatric epilepsy is a common neurological disorder with a prevalence rate of 4 - 6 per 1,000 children1. It can have a major impact on children&#39;s development2 and may significantly affect their adult life. Long-term follow-up studies in childhood-onset epilepsy indicate that approximately 30% of patients with epilepsy become medically intractable3-6, and usually require resective epileptic surgery. In many of these patients, epilepsy surgery leads to significant reduction in seizure frequency and often to seizure freedom. To be successful, epilepsy surgery should achieve a seizure-free state with minimal or no functional deficits. This requires careful delineation of the Epileptogenic Zone (EZ)7, the &#39;area of cortex that is indispensable for the generation of epileptic seizures&#39;8. The EZ cannot be measured directly; its location is estimated based on concordant data from a multitude of tests that identify other cortical zones. Invasive intracranial electroencephalography (iEEG) serves as the gold standard for the localization of the seizure onset zone (SOZ), the region where seizures are generated and originate on ictal recordings. However iEEG is costly, reliant on cooperation of the child, carries some risk for infection and bleeding9, and may induce additional neurological damage during the implantation10. Furthermore, the recordings may lead to erroneous conclusions since large areas of the brain are left unexplored. Thus, a robust presurgical biomarker that helps in the identification of the EZ is needed to the success of surgical epilepsy treatment.
Pathological HFOs&#160;(80 - 500 Hz)11,12 have emerged over the last decade as a biomarker for the identification of the epileptogenic tissue that may improve the presurgical diagnosis and surgical outcome of patients with epilepsy13. Reports using microelectrodes combined with depth EEG&#160;electrodes showed the presence of HFOs in patients with epilepsy. HFOs were also found using standard macroelectrodes during the ictal and interictal periods. Recent studies have shown that HFOs identify the SOZ with higher sensitivity and specificity compared to the irritative zone14,15, the zone that generates the IEDs, and that the surgical removal of the HFO-generating tissue correlates with better outcomes than the removal of the SOZ or the irritative zone15. HFOs are commonly categorized as ripples (80 - 250 Hz) or fast ripples (250 - 500 Hz). Fast ripples have been more closely linked to pathological activity and to the localization of the SOZ16, but investigations of human intracranial recordings indicate that both ripples and fast ripples increase in epileptogenic regions17.
Despite these promising findings, HFOs are not yet suited for the diagnosis or monitoring of epilepsy in clinical practice. Primary barriers remain: (i) the lack of a formal and global definition for HFOs; (ii) the consequent heterogeneity of the methodological approaches used for their study; and (iii) the practical difficulties to detect and localize them noninvasively from scalp recordings. The latter stems from the fact that electrodes are far away from the source of the signal, the signal might be blurred by background noise and muscle activity, and the signal might be distorted by the scalp or the fontanels and sutures in the skull, especially in infant patients. Moreover, it is difficult to distinguish between normal and abnormal HFOs18,19 since both ripples and fast ripples are present even in normal human brain tissue20. Early studies reported HFOs in scalp EEG in only a small (0.2&#160;- 3.4%) portion of patients with epilepsy21-23. However, recent studies have shown that HFOs can be detected noninvasively with scalp EEG. Ictally, HFOs have been reported at the onset of epileptic spasms in children (50 - 100 Hz24, 40 - 120 Hz25), as well as at the onset of tonic seizures in Lennox-Gastaut syndrome (50 - 100 Hz)26. Interictal HFOs (70 - 200 Hz) were first observed on scalp EEG in children with sleep-induced electrical status epilepticus27. Then, interictal HFOs (80 - 200 Hz) were identified in the scalp EEG of patients with focal epilepsy with higher rates inside the SOZ28. Interestingly, HFOs were more frequent in patients with high numbers of interictal epileptiform discharges (IEDs), and they were found to be more specific than IEDs for the SOZ29, highlighting the relation of HFOs with epileptogenicity.
MEG&#160;seems to present significant advantages compared to scalp EEG for the noninvasive detection and localization of HFOs: (i) high frequency activity in MEG is less susceptible than EEG to contamination from muscular activity30-31, (ii) MEG signals are not distorted by skull conductivity and less distorted than EEG by unfused regions of the cranial bone such as fontanel or suture, and (iii) MEG sensor arrays have higher density compared to EEG that always faces the problem of salt bridges between electrodes when the head is small, as with children. Evidence from phantom constructions that simulate HFOs generators suggested that HFOs can be detected and localized with high localization accuracy (2 - 3 mm) with MEG32. Several recent studies reported HFOs in the MEG signals recorded from patients with epilepsy in the ripple frequency band33-38. Time-frequency analysis has shown that MEG data contain high frequency components related to the EZ33-36. However, only a few studies have identified interictal HFOs as visible events standing out of the background signal in the time domain, as typically done with iEEG37-38. Van Klink et al.37 detected HFOs in the ripple band using virtual channels constructed with beamforming techniques based on spatial information obtained from IEDs. Von Ellenrieder et al.38 detected HFOs in MEG signals from the physical sensors independently of the IEDs and used the Maximum Entropy on the Mean (MEM) method to localize their sources and to investigate their correlation with the EZ. Rampp et al. (2010) also detected epileptic high gamma oscillations with MEG, which were spike-locked or spike-independent, and localized this activity with minimum-norm source analysis39. They found that characteristics of these fast oscillations (i.e., clear onset of full-band average and maximum amplitude of oscillations) were highly associated with the SOZ. HFOs were also detected with MEG during ictal activity in pediatric patients with epileptic spasms40. However, MEG presents some distinct limitations compared to scalp EEG: (i) it is insensitive to sources that have a radial orientation with respect to the center of the head, (ii) it does not allow long recordings that increase the possibility to detect and record ictal events, and (iii) its sensors cannot conform to the shape of head of each individual since the helmet and sensor array within the helmet are all fixed in shape. Thus, the ideal setup that maximizes the possibility to detect and localize the epileptogenic activity is by combining information from both scalp EEG and MEG.
In this study, we aim to illustrate the methodology we follow for the noninvasive detection of interictal HFOs by using simultaneous recordings of scalp EEG and MEG from pediatric patients with medically refractory epilepsy. We present the setup of the recordings and the pipeline of data analysis using a semi-automated method that we have developed for the detection of HFO events in simultaneous MEG and EEG data. Finally, we also present the localization of the underlying generators of scalp HFOs, obtained by solving the inverse problem, and compare it with the SOZ as this was defined by the epileptologists.

<table border="0" cellpadding="0" cellspacing="0" width="1028">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">VectorView MEG system</td>
			<td style="width:287px;">Elekta-Neuromag, Finland</td>
			<td style="width:119px;"></td>
			<td style="width:407px;">MEG System</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magentically Shielded Room</td>
			<td style="width:287px;">Imedco, Hagendorf, Switzerland</td>
			<td style="width:119px;"></td>
			<td style="width:407px;">Three-layer MSR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EEG system</td>
			<td style="width:287px;">Elekta-Neuromag, Finland</td>
			<td style="width:119px;"></td>
			<td>70 Channel EEG system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3D digitizer</td>
			<td style="width:287px;">Polhemus, Colchester, VT</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

interictal high frequency oscillations detected with simultaneous magnetoencephalography and electroencephalography as biomarker of pediatric epilepsy

Crucial to the success of epilepsy surgery is the availability of a robust biomarker that identifies the Epileptogenic Zone (EZ). High Frequency Oscillations (HFOs) have emerged as potential presurgical biomarkers for the identification of the EZ in addition to Interictal Epileptiform Discharges (IEDs) and ictal activity. Although they are promising to localize the EZ, they are not yet suited for the diagnosis or monitoring of epilepsy in clinical practice. Primary barriers remain: the lack of a formal and global definition for HFOs; the consequent heterogeneity of methodological approaches used for their study; and the practical difficulties to detect and localize them noninvasively from scalp recordings. Here, we present a methodology for the recording, detection, and localization of interictal HFOs from pediatric patients with refractory epilepsy. We report representative data of HFOs detected noninvasively from interictal scalp EEG and MEG from two children undergoing surgery.
The underlying generators of HFOs were localized by solving the inverse problem and their localization was compared to the Seizure Onset Zone (SOZ) as this was defined by the epileptologists. For both patients, Interictal Epileptogenic Discharges (IEDs) and HFOs were localized with source imaging at concordant locations. For one patient, intracranial EEG (iEEG) data were also available. For this patient, we found that the HFOs localization was concordant between noninvasive and invasive methods. The comparison of iEEG with the results from scalp recordings served to validate these findings. To our best knowledge, this is the first study that presents the source localization of scalp HFOs from simultaneous EEG and MEG recordings comparing the results with invasive recordings. These findings suggest that HFOs can be reliably detected and localized noninvasively with scalp EEG and MEG. We conclude that the noninvasive localization of interictal HFOs could significantly improve the presurgical evaluation for pediatric patients with epilepsy.

Pediatric patients with refractory epilepsy were recruited from the Epilepsy Clinic of Boston Children&#39;s Hospital (Boston, USA). Here, representative data from 2 patients are presented: a 15-year-old girl with encephalomalacia of the right middle cerebral artery (MCA) region (patient 1), and an 11-year-old boy with left parietal/superior temporal encephalomalacia (patient 2). The data were collected as part of their presurgical workup for surgery. Simultaneous MEG and EEG recordings were performed at the Athinoula A. Martinos Center for Biomedical Imaging.
For both patients, HFOs were identified in the ripple frequency band (80 - 150 Hz) occurring in both EEG and MEG and overlying IEDs. Figure 4 presents 10 s of simultaneous scalp EEG and MEG data with interictal activity (upper panels) from patient 1. The same figure also reports an extended time scale portion (2 s) of the signals, which shows the detected HFOs in the time domain (middle panels) and in the time-frequency plane (lower panel). This patient showed a rate of 8.8 HFOs/min. For clarity, the display of the time-frequency plane is restricted from 80 - 150 Hz because no prominent activity was observed for frequencies above 150 Hz. In patient 1, the automatic detection identified 248 HFOs events within 8.65 min of recordings. After visual review of the detected events, 76 events were kept that were considered to be real HFOs, obtaining a rate of 8.8 HFOs/min.
<img alt="Figure 4" src="/files/ftp_upload/54883/54883fig4.jpg" /> 
Figure 4: HFOs Overlaid on IEDs Detected from Scalp EEG and MEG. Upper panel: IEDs from patient 1 detected from scalp EEG (left) and MEG (right). 10 s of data filtered from 1 - 70 Hz. Middle panel: Extended time scale display (2 s) of the EEG and MEG data highlighted with grey background in the upper panel. Data are filtered from 80 - 150 Hz. Lower panel: Time-frequency plane of 2 representative EEG and MEG channels. <a href="http://cloudflare.jove.com/files/ftp_upload/54883/54883fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 5 presents the HFOs localization results for patient 1 from both scalp EEG and MEG. Both techniques localized the HFOs at the vicinity of the lesion in a location close to the right temporo-parietal junction. MEG and EEG presented slightly different localization results: MEG activity was located more anteriorly compared to EEG and closer to the posterior boundary of the lesion. The distance between the two maxima of wMEM activity for the MEG and EEG was 15.0 mm. HFOs localization was also adjacent to the irritative zone as this was defined by MEG. The Euclidian distance between the averaged ECDs and the maximum wMEM activity was 18.7 mm for the MEG and 28.0 mm for the scalp EEG. For patient 2, we identified 8 min of data during slow-wave non-REM sleep with IED and minimal artifacts that we used for the HFO analysis.
The number of scalp HFOs was significantly lower than in patient 1 showing a rate of 0.4 HFOs/min. Figure 6 presents the source localization results of scalp HFOs for MEG and scalp EEG, the IEDs localized by MEG using ECDs, and the location of HFOs detected by iEEG. The distance between the 2 maxima of wMEM activity for the MEG and EEG was 16.4 mm. The distance between the averaged ECDs and the maximum wMEM activity was 10.9 mm for the MEG and 24.1 mm for the EEG. The HFO zone identified noninvasively by scalp EEG and MEG was at the same location where the HFOs were identified invasively with iEEG. The electrodes with the highest number of HFOs were LA51, LA52, and LA53, as shown in Figure 6, whose localization was concordant with the HFO activity localized noninvasively.
<img alt="Figure 5" src="/files/ftp_upload/54883/54883fig5.jpg" /> 
Figure 5: Localization of HFOs and IEDs. HFOs zone localized by using the wMEM with MEG and EEG from patient 1 overlaid on patient&#39;s MRI (left). The HFOs map represents the average of the normalized activation values thresholded at 60% of the maximum activity. IEDs localized by ECDs with MEG (right). The locations and orientations of the ECDs are indicated in cyan. The purple triangle indicates the location of the averaged ECDs (46 ECDs), and the red rectangle the location of the ECD from averaged IEDs (28 IEDs). For the averaging of the IEDs, we identified spikes with similar morphology. <a href="http://cloudflare.jove.com/files/ftp_upload/54883/54883fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54883/54883fig6.jpg" /> 
Figure 6: Spatial Concordance between the HFO Zone Localized Noninvasively with Scalp EEG and MEG, the Irritative Zone, and the HFO Zone Localized Invasively with iEEG. Upper panels: Localization of HFOs from patient 2 with scalp EEG (left) and MEG (middle), and localization of IEDs with MEG (right) overlaid on patient&#39;s MRI. The HFOs map represents the average of the normalized activation values thresholded at 60% of the maximum activity. Cyan circles and bars indicate the locations and orientations of the ECDs. The purple triangle indicates the location of the averaged ECDs (30 ECDs), and the red rectangle the location of the ECD from averaged IEDs (21 IEDs). Lower panels: Localization of HFOs on iEEG (left) and 2 s&#160;of recordings from channels with the highest HFO rate. Implanted electrodes are shown on the patient&#39;s MRI. The electrode location is obtained by co-registering the post-implanted computed tomography (CT) and MR images. The electrodes with the highest rate of HFOs are highlighted on the map of the grid implanted on the patient&#39;s cortex. <a href="http://cloudflare.jove.com/files/ftp_upload/54883/54883fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The representative results presented here indicate the potential use of the described technique in the clinical setting for the noninvasive identification and localization of HFOs during the pre-surgical evaluation of pediatric patients with medically refractory epilepsy. For both patients, HFOs were localized concordant for scalp EEG and MEG (see Figure 5). Localization differences of few mm may be attributed to the superior localization ability of high-density MEG compared to scalp EEG70, or may represent different underlying generators71. The localization was also concordant with the irritative zone as this was defined by MEG (see Figure 5 - right panel). In patient 2, data from iEEG were also available. The HFOs zone localized noninvasively with scalp EEG and MEG was concordant with the HFOs zone defined invasively with iEEG (see Figure 6). The outcomes of the proposed method can guide the placement of grids during epilepsy surgery for the potential identification of the EZ.

Watch this Scientific Journal Video about Interictal High Frequency Oscillations Detected with Simultaneous Magnetoencephalography and Electroencephalography as Biomarker of Pediatric Epilepsy at JoVE

Interictal High Frequency Oscillations Detected with Simultaneous Magnetoencephalography and Electroencephalography as Biomarker of Pediatric Epilepsy

High Frequency Oscillations (HFOs) have emerged as presurgical biomarkers for the identification of the epileptogenic zone in pediatric patients with medically refractory epilepsy. A methodology for the noninvasive recording, detection, and localization of HFOs with simultaneous scalp electroencephalography (EEG) and magnetoencephalography (MEG) is presented.

Crucial to the success of epilepsy surgery is the availability of a robust biomarker that identifies the Epileptogenic Zone (EZ). High Frequency ...

interictal-high-frequency-oscillations-detected-with-simultaneous

Research

Education

JoVE Core

JoVE Journal

Electrical Engineering

Medicine

Unit 1

Linear Time- Invariant Systems

SCIENTISTS IN ACTION

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

Surgical Management of Meatal Stenosis with Meatoplasty

Meatal stenosis is a common urologic complication after circumcision. Children present to their primary care physicians with complaints of deviated urinary stream, difficult-to-aim, painful urination, and urinary frequency. Clinical exam reveals a pinpoint meatus and if the child is asked to urinate, he will usually have an upward, thin, occasionally forceful urinary stream with incomplete bladder emptying. The mainstay of management is meatoplasty (reconstruction of the distal urethra /meatus). This educational video will demonstrate how this is performed.

Meatoplasty, surgical management of meatal stenosis.

Meatal stenosis is a common urologic complication after circumcision.  Children present to their primary care physicians with complaints of deviated ...

Guidelines for Elective Pediatric Fiberoptic Intubation

Fiberoptic intubation in pediatric patients is often required especially in difficult airways of syndromic patients i.e. Pierre Robin Syndrome. Small babies will desaturate very quickly if ventilation is interrupted mainly to high metabolic rate. We describe guidelines to perform a safe fiberoptic intubation while maintaining spontaneous breathing throughout the procedure. Steps requiring the use of propofol pump, fentanyl, glycopyrrolate, red rubber catheter, metal insuflation hook, afrin, lubricant and lidocaine spray are shown.

We describe guidelines to perform a safe and efficient elective fiberoptic intubation in pediatric patients while maintaining spontaneous ventilation.

Fiberoptic intubation in pediatric patients is often required especially in difficult airways of syndromic patients i.e. Pierre Robin Syndrome. Small ...

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

Brain Source Imaging in Preclinical Rat Models of Focal Epilepsy using High-Resolution EEG Recordings

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with focal epilepsy. This methodology relies on the assumption that seizures originate from the same regions of the brain from which interictal epileptiform discharges (IEDs) emerge. Preclinical models are very useful to find correlates between IED locations and the actual regions underlying seizure initiation in focal epilepsy. Rats have been commonly used in preclinical studies of epilepsy1; hence, there exist a large variety of models for focal epilepsy in this particular species. However, it is challenging to record multichannel EEG and to perform brain source imaging in such a small animal. To overcome this issue, we combine a patented-technology to obtain 32-channel EEG recordings from rodents2 and an MRI probabilistic atlas for brain anatomical structures in Wistar rats to perform brain source imaging. In this video, we introduce the procedures to acquire multichannel EEG from Wistar rats with focal cortical dysplasia, and describe the steps both to define the volume conductor model from the MRI atlas and to uniquely determine the IEDs. Finally, we validate the whole methodology by obtaining brain source images of IEDs and compare them with those obtained at different time frames during the seizure onset.

This video introduces the preparation, recording, and source analysis procedures of high-resolution EEG on sedated rats with a particular preclinical model of focal epilepsy under noninvasive conditions.

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

A Pipeline for 3D Multimodality Image Integration and Computer-assisted Planning in Epilepsy Surgery

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality image integration can be technically demanding, and is underutilised in clinical practice. We have developed a single software platform for image integration, 3D visualization and surgical planning. Here, our pipeline is described in step-by-step fashion, starting with image acquisition, proceeding through image co-registration, manual segmentation, brain and vessel extraction, 3D visualization and manual planning of stereoEEG (SEEG) implantations. With dissemination of the software this pipeline can be reproduced in other centres, allowing other groups to benefit from 3DMMI. We also describe the use of an automated, multi-trajectory planner to generate stereoEEG implantation plans. Preliminary studies suggest this is a rapid, safe and efficacious adjunct for planning SEEG implantations. Finally, a simple solution for the export of plans and models to commercial neuronavigation systems for implementation of plans in the operating theater is described. This software is a valuable tool that can support clinical decision making throughout the epilepsy surgery pathway.

We describe the steps to use our custom designed software for image integration, visualization and planning in epilepsy surgery.

Epilepsy surgery is challenging and the use of 3D multimodality image integration (3DMMI) to aid presurgical planning is well-established. Multimodality ...

A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations over time in the blood-oxygenation level dependent signal. rs-fcMRI has been applied extensively to identify abnormalities in brain connectivity in different neurologic and psychiatric diseases. However, the relationship among rs-fcMRI connectivity abnormalities, brain electrophysiology and disease state is unknown, in part because the causal significance of alterations in functional connectivity in disease pathophysiology has not been established. Transcranial Magnetic Stimulation (TMS) is a technique that uses electromagnetic induction to noninvasively produce focal changes in cortical activity. When combined with electroencephalography (EEG), TMS can be used to assess the brain's response to external perturbations. Here we provide a protocol for combining rs-fcMRI, TMS and EEG to assess the physiologic significance of alterations in functional connectivity in patients with neuropsychiatric disease. We provide representative results from a previously published study in which rs-fcMRI was used to identify regions with abnormal connectivity in patients with epilepsy due to a malformation of cortical development, periventricular nodular heterotopia (PNH). Stimulation in patients with epilepsy resulted in abnormal TMS-evoked EEG activity relative to stimulation of the same sites in matched healthy control patients, with an abnormal increase in the late component of the TMS-evoked potential, consistent with cortical hyperexcitability. This abnormality was specific to regions with abnormal resting-state functional connectivity. Electrical source analysis in a subject with previously recorded seizures demonstrated that the origin of the abnormal TMS-evoked activity co-localized with the seizure-onset zone, suggesting the presence of an epileptogenic circuit. These results demonstrate how rs-fcMRI, TMS and EEG can be utilized together to identify and understand the physiological significance of abnormal brain connectivity in human diseases.

Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Topical Airway Anesthesia for Awake-endoscopic Intubation Using the Spray-as-you-go Technique with High Oxygen Flow

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can jeopardize their spontaneous breathing, topical anesthesia of the airway is a popular technique. The spray-as-you-go technique represents one of the simplest opportunities to anesthetize the airway mucosa. The application of local anesthetic through the working channel of the flexible endoscope is a widespread practice for anesthetists as well as pulmonologists. There is neither need for additional devices nor special training as a pre-requisite to perform this technique. However, a known clinical problem is the coughing and gagging reflex that may occur when the liquid anesthetic strikes the airway mucosa and other sensitive structures like the vocal cords. This can be avoided by the use of oxygen applied through the working channel with the aim of fogging the local anesthetic into finer particles. Furthermore, the oxygen flow provides a higher oxygen supply and contributes to a better view, dispersing mucus secretions and blood away from the lens. Using an atomizer with a high oxygen flow of 10 L/min we maximized these benefits, caused less coughing and had more satisfied and therefore cooperative patients. Possible, but very rare complications of using oxygen flow including gastric insufflation, organ rupture or barotrauma did not arise. We attribute the complication-free use of high oxygen flow to the design of the set, which permits flow and pressure release.

The topical anesthetic lidocaine was atomized using a high oxygen flow through the working channel of a flexible intubating endoscope to achieve topical airway anesthesia for awake endotracheal intubation. We prefer this modified spray-as-you-go technique for endoscopic intubation to classical bolus application because of higher patient satisfaction and better compliance.

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can ...