# Early Signatures of Brain Injury in the Preterm Neonatal EEG
## 2 Materials and Methods
### Experimental Protocols  
In this study, a total of seven preterm Romney/Suffolk fetal sheep at the gestational ages of 103 \(\pm\) 1 days were used (full term gestation: 145 days). The surgical procedures were conducted using aseptic techniques, as previously described [10]. Prior to surgery, the ewes were fasted for 18 h, with access to water. They were administered long-acting oxytetracycline (20 mg/kg, Phoenix Pharm, Auckland, New Zealand) intramuscularly 30 min before the start of surgery. Anesthesia was induced using intravenous propofol (5 mg/kg, AstraZeneca Limited, Auckland, New Zealand) and maintained with 2-3% isoflurane in oxygen. Throughout the procedure, the depth of anesthesia, maternal heart rate, and respiration were closely monitored by trained staff. To maintain fluid balance, the ewes received a continuous infusion of isotonic saline (at approximately 250 mL/h).  
Following a midline abdominal incision, the fetuses were exposed and an inflatable silicone occluder (OC16 HD, 16 mm, In Vivo Metric, Healdsburg, CA, USA) was loosely placed around the umbilical cord to allow for post-surgical occlusion, inducing fetal hypoxic-ischemic (HI) injury [6,8]. EEG electrodes, consisting of two pairs of electrodes made from a 7-stranded stainless-steel wire (AS633-7 SSF; Cooner Wire Co.: Los Angeles, CA, USA), were placed on the dura over the parasagittal parietal cortex. A reference electrode was sewn over the occupit. Additional instruments, such as thermistors for temperature measurement, were placed accordingly (data not used in this study). The surgical incisions were closed, and antibiotics (80 mg Gentamicin, Pharmacia and Upjohn, Rydalmere, New South Wales, Australia) were administered into the amniotic sac. Post-operative care included housing the sheep together in separate metabolic cages with ad libitum access to food and water. The room temperature was maintained at 16 \(\pm\) 1 \({}^{\circ}\)C, with a humidity level of 50 \(\pm\) 10%, and a 12 h light/dark cycle (lights on at 06:00 h).  
Sheep received intravenous antibiotics daily for four days (600 mg benzylpencillin sodium, Novartis Ltd., Auckland, New Zealand, and 80 mg gentamicin) to ensure post-operative maternal care and minimize the risk of infection. The patency of fetal catheters was maintained by continuous infusion of heparinized saline, while the maternal catheter was flushed daily to maintain patency.  
Data were recorded and saved continuously at 1024 Hz sampling frequency for off-line analysis using custom data acquisition programs (LabView for Windows, National Instruments, Austin, TX, USA). The raw EEG recordings were initially low-pass filtered by using a 6 th order anti-aliasing Butterworth filter with a cut-off set at 500 Hz. The signal was then amplified by a gain \(\times\)10,000 and then high-pass filtered by a first-order filter with a cut-off frequency set at 1.6 Hz. The recordings were then digitized at 4096 Hz, low-pass filtered using a 10 th-order low-pass inverse Chebyshev at 128 Hz (in software), and then decimated down to 1024 Hz sample rate, before saving to file. Data from the last stage were recorded for 72 h and then decoded into Matlab for analysis. Here, we used the experimental EEG recordings during the first 6 h after HI.