The overall aim of the following experiment is to record and characterize human infant nociceptive specific brain and spinal cord activity. This is achieved using EEG and EMG techniques to measure electrophysiological activity in the central nervous system. Following clinically essential noxious procedures, the first step is to ensure that high quality physiological recordings are obtained from infants when they experience experimental, tactile, and essential noxious stimuli.
Next, post-processing analytical techniques need to be applied to the data in order to characterize the evoked patterns of activity. Results can be obtained that show no susceptive specific brain and spinal cord activity can be recorded from the human infant brain. This method will help us understand how the developing nervous system responds to noxious stimulation.
To begin set up for this experiment, first prep the infant's skin and then place a minimum of 16 individual disposable EEG silver, silver chloride cup electrodes on the head. According to the modified international 10 20 electrode placement system, use EEG conductive paste to optimize electrode skin electric coupling. Here a schematic of electrode placement for EEG recordings can be seen modified from the international 10 20 electrode placement system.
Use FCZ as the reference electrode for the recording. Use the same ground electrode for the ECG and the EEG. Place a ground electrode onto the chest or head.
Then to set up the ECG recording prep the skin and place ECG electrodes on the left and right side of the chest. Tie the electrode leads together to minimize electrical interference. Next place a movement transducer on the abdomen to measure respiration.
The next step is to prep the skin and place EMG electrodes on the biceps for morice of both legs. Now place a pulse oximeter probe on the foot contralateral to the foot that will be stimulated, and make sure that the probe is secured in place. Check the monitor for the EEG signal and check the oxygen saturation and heart rate are recorded without signal dropouts.
Finally, set up a tripod mounted camcorder to frame the face of the infant so that changes in facial expression can be recorded. Place a light emitting diode LED in the camera frame. The LED is linked to the timing circuit so that it will flash when stimulation is presented to synchronize the E-E-G-E-M-G and video recording.
Once the setup is complete, begin data collection. Start the video recording and after the infant is settled, hold the foot as if performing a heel lance and event mark the EEG and EMG recordings. This epoch will be used to identify a section of the background control.
Next, apply touch stimulation by lightly tapping a rubber bung against the heel. Stimulate the foot that is not attached to the pulse oximeter. Here the touch stimulation is event marked by using a rubber bung attached to an impedance head on a tendon hammer, which is electronically linked to the recording equipment.
The video recording is event marked by the LED flash. Repeated touches may be applied and the stimulus can be applied to different regions of the body. IE the shoulder.
Now apply control stimulation by rotating the lancet by 90 degrees and placing it against the foot so that when the spring loaded blade is released, it does not contact the skin. After the EEG activity is settled, perform the clinically essential heel lance in accordance with clinical practice as a heel. Lance was not performed on the infant filmed up to this point shown here is a heel lance on a different infant time locking of the heel.
Lance should be performed as it was for the control stimulation following the heel Lance, do not squeeze the foot for at least 30 seconds to ensure that the recorded responses are solely due to the lance. After collecting the required quantity of blood, prepare the samples for clinical analysis. Save the data and stop all the recording equipment.
Then remove the electrodes. Finally, record the infant's demographic information and the experimental details. Input this data into an anonymized database for safe storage and future reference.
Repeat this procedure in the required sample of infants in the study. To begin EEG data analysis, first, create EG epoch of 1.7 seconds that correspond to each touch control and lance stimulation and the background EEG. These epoch should start 0.6 seconds before each event.
The number of epochs corresponding to each modality should be the same baseline, correct the epoch by subtracting the mean baseline signal. Then high pass, filter them at 0.1 hertz. Consider the epoch recorded at CPZ or CZ for further analysis and exclude epochs that were contaminated by movement artifact with a change in amplitude greater than 50, microvolts in less than 50 milliseconds.
Repeat this for all recordings. Next, align the traces recorded from each infant to correct for latency jitter between 50 and 30 milliseconds. Post stimulation.
Conduct principle component analysis in this time interval to identify the tactile potential being the EEG activity related to the tactile stimulation. Consider the EPOCH to be the variables and the time points. The observations.
Principle component analysis. Decomposes the EEG epochs into basic wave forms, termed principle components or PCs, and represents systematic variation in the amplitude of the signal across time points. Now align the traces to correct for latency jitter between 300 and 700 millisecond post stimulation and conduct principle component analysis in this time interval For the EMG data analysis, first, calculate the root mean square of the EMG signal in the first 1000 milliseconds post stimulation for the control and lance stimuli.
Then do a T-test on the root mean square values to determine the nociceptive specific spinal reflex withdrawal. Here we see the grand average at CZ obtained across all stimulation types after alignment between 50 and 300 milliseconds. The principle components in bold line represents a sensory potential evoked by both the tactile and noxious stimulation because the weights of this component are significantly larger following the tactile and noxious stimulation compared to the background EEG.
In contrast, the principle component obtained between 300 and 700 milliseconds after the stimulus onset represents a nociceptive specific potential. The weight of this component is significantly larger following the noxious stimulation compared to the tactile stimulation and background. EEG displayed Here are examples of the sensory potential in blue at cz evoked by tactile stimulation in three infants, and here are examples of the nociceptive specific potential in green at cz evoked by noxious lance in three infants.
Finally, here we see an example of EMG activity in an infant after noxious heel lance and non noxious touch stimulation of the heel. The root means square EMG activity is significantly larger following noxious stimulation compared with the non noxious. This technique will pave the way in the field of neuroscience for researchers to understand the development of pain processing.
