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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Preparation
- Make sure the baby is settled. It should be quiet and settled, without sucking motions during the recording because of movement artifacts. The baby can be asleep.
- Measure the neonate's head circumference with a measuring tape to define the size of the electroencephalography (EEG) cap.
- Identify the active electrode position: Cz / vertex position by marking the middle point between nasion and inion and the middle point between the left and right preauricular points with a skin marker pencil. For positioning the electrodes, see also Figure 1.
- Identify the positions of the ground electrode: right forehead (Fp2), and the reference electrode: left mastoid (A1).
- Clean the electrode sites (Cz, Fp2, and A1) with disinfectant using a cotton swab. In case of a lot of hair, separate the hair to visualize the scalp.
- Gently scrub the electrode sites with EEG prepping paste, using a cotton swab, to lower the impedance.
- Place the EEG cap with the electrodes attached on the neonate's head.
- Inject conductive EEG gel into the electrodes using a syringe with a short plastic needle to optimize the contact between the electrodes and the scalp.
- Adjust the electrodes until the impedance is below 50 kΩ as described in the manufacturer's recommendations and published guidelines.
- Position a camera to record the neonate's facial expressions.
- Connect the flat-tip probe to the contact trigger device, which is fixed to the EEG recording device. When the flat-tip probe reaches the nominal force on the skin, a trigger signal is generated by the contact trigger device. This signal is sent to the computer, tagging the EEG recording with a trigger mark.
2. Measurement
- Select a study name to store the data.
- Add online bandpass filters:
- Display filters:
Low cutoff filter: Frequency: 1 Hz
High cutoff filter: Frequency: 70 Hz
Notch filter: 50 Hz
Sampling rate: 2000 Hz
- Start the EEG and video recording.
- Hold the neonate's right hand in a horizontal position. The stimulus can also be applied on a different site, e.g. the foot, which will result in longer latency than from the hand, please see for details 3.7.
- First, record background EEG activity. While the neonate's hand is held, manually annotate the EEG recording to record periods where no stimuli are applied, and the infant is resting.
- Conduct the required amount of flat-tip probe stimuli according to the study design on the neonate's right hand. In our experimental design, set the number of flat-tip probe stimuli at 50. Be careful to use the flat-tip probe perpendicularly to the neonate's hand so the tip does not bend, and the correct force is applied. Perform the flat-tip probe stimuli using a minimum of a 2 to 3 s inter-stimulus interval (ISI) to avoid summation.
- Stop all the recordings.
- Document the experimental setting details.
3. Data Analysis
- Filter the raw EEG data offline using a high-pass filter at 1 Hz and a low-pass filter at 30 Hz.
- Segment the data in epochs of 1,500 ms (500 ms before to 1,000 ms after stimulus onset).
- Perform a baseline correction to the pre-stimulus interval.
- Manually reject the EEG epochs containing artifacts, such as movement artifacts and noise, after visual inspection.
- Average the EEG epochs (for the background and stimulus response separately).
- Woody filter the data with a maximum jitter of ± 50 ms in the time window 0 - 1,000 ms post stimulus onset. This allows for latency differences between the infants. Do this for the background and stimulus responses separately.
- Project a template of noxious-evoked brain activity onto the data to ascertain the magnitude of the noxious-evoked response within each individual trial. The template describes a characteristic waveform of the noxious-evoked response, and the magnitude reflects the amount of this noxious-evoked response within the individual trial.
NOTE: The time window on which to project the template is dependent on the age of the infant and the stimulus position. If stimuli are applied to the hand, then the time window of interest is 200 - 500 ms post stimulus. If the stimuli are applied to the foot, then the time window of interest is 400 - 700 ms post stimulus. Note that this template has only been validated for infants between 34 - 43 weeks gestation.
- Check that the magnitude of the noxious-evoked brain activity calculated using the template is significantly higher following the stimulus compared with in the background EEG.
- Analyze the behavioral facial expression afterwards (the camera is linked to the EEG recording) using the Neonatal Facial Coding System.
