May 5, 2020 Dear Dr. DSouza, Please find enclosed revisions on our manuscript entitled “Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice” (JoVE61346). We thank the reviewers and editors for their time and thoughtful review. Please see our response to reviewers letter below and the attached revisions in the manuscript (with tracked changes in MS Word document). This manuscript is being submitted only to Journal of Visualized Experiments and will not be submitted elsewhere while under consideration. Further, it has not been published elsewhere in whole or in part.	All authors are responsible for reported research and have participated in the concept and design, analysis and interpretation of data, and/or drafting or revising of the manuscript. They have approved the manuscript as submitted. We have no conflicts of interest other than NIH funding to Dr. Kapur and myself. Sincerely, Jennifer Burnsed, MD, MS Assistant Professor of Pediatrics and Neurology Division of Neonatology University of Virginia PO Box 800386 Charlottesville, VA 22908 jcw5b@virginia.edu Phone: 434-924-5428 Fax: 434-243-0649

Division of Neonatology

Robert A. Sinkin, MD, MPH Division Head rsinkin@virginia.edu Jennifer C. Burnsed, MD, MSc jcw5b@virginia.edu Karen D. Fairchild, MD kdf2n@virginia.edu David A. Kaufman, MD dak4r@virginia.edu Sarah Miller, DO sem5bj@virginia.edu Peter Murray, MD, MSM pm7dn@virginia.edu Alix O. Paget-Brown, MD aof4e@virginia.edu Brynne A. Sullivan, MD bsa4m@virginia.edu Jonathan R. Swanson, MD, MSc jrs3yc@virginia.edu Brooke D. Vergales, MD bld5j@virginia.edu Santina A. Zanelli, MD, MSc sz5d@virginia.edu ------------------ John Kattwinkel, MD jk3f@virginia.edu Professor Emeritus Professor of Research in Pediatrics Robert J. Boyle, MD rjb6j@virginia.edu Professor Emeritus Administration Linda Crawford Steve Garrison Tammy Mawyer Research Coordinators Monika Thielen Joey Michel

Response to Reviewers

Reviewer #1:
Manuscript Summary:
The authors of "Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice" offer a detailed protocol for implantation of an electrode into the brain (hippocampus, parietal cortex) to measure seizure EEG in mouse pups. The implantation surgery is described, and conducted 24 hours prior to carotid artery ligation and hypoxia to produce brain injury. This injury procedure is based on the common and accepted Rice Vannucci model. Pups are subjected to HI at postnatal day 10. This is considered a model of term birth. The demonstrated that EEG can be measured continuously during hypoxia, and for at least 2 hours post hypoxia. The nature of the seizures tends to change after hypoxia, from largely constrained to the ipsilateral hemisphere to both hemispheres. The authors also describe the behavioral indicators of seizures and note that the EEG can be matched to behavior using their dual video/EEG data acquisition set up. The authors carefully list vendor catalog numbers and various details to promote success of others that might chose to use this technique. Overall, the methods are clear, and since this technique is new to this reviewer, but the HI procedure is not, I assume that the EEG protocol is as detailed at the HI protocol. Indeed, all critical details are present in the HI protocol. The authors make the important note that the pups should be returned to the dam in a group after electrode implantation. I am eager to see this published in video format.
Major Concerns:
No major concerns
Minor Concerns:
No minor concerns
Reviewer #2:
Manuscript Summary:
The authors describe a method for monitoring video/EEG from neonatal mouse pups immediately before, during and immediately after hypoxia/ischemia. They provide a clear description of the method which is placed into context with regard to neonatal stroke. The method provides a potential means to understand neuropathological mechanisms associated with neonatal hypoxia/ischemia. There are a couple of concerns with the manuscript:
Major Concerns:
1) It's useful to have baseline EEG before hypoxia as a control as indicated. However, it would be helpful to also see results from sham operated controls to define the impact of electrode placement and carotid surgery without ligation.

For each mouse we have performed a minimum of 30 minutes of baseline (pre-injury) EEG recording so that each mouse serves as its own control. This allows us to control for any impact of electrode placement. Recordings noted to have excessive artifact or other issues with recording are excluded. In addition, gross brains are examined following the experiment to verify correct electrode placement.

2) It’s not clear if EEG recording is conducted before ligation of the carotid artery. This should be clarified.

Baseline recording is conducted for 30 minutes prior to ligation of the carotid artery. Please see lines 126-127 of the protocol.

3) Is occlusion of one carotid adequate to induce ischemia? Do pups have an underdeveloped circle of Willis? Does the contralateral carotid not provide adequate collateral flow?

Yes, this is adequate to induce ischemia. This is a well-established model of neonatal hypoxic ischemic injury which has been used in this field for over 30 years and results in a significant ischemic injury which has been thoroughly described in numerous studies (Rice 1981, Burnsed 2015, Ferriero 2019).
Minor Concerns:
1) in section 4.3 please indicate where the incision is being made.

This has been clarified, please see line 135.
Reviewer #3:
Manuscript Summary:
The manuscript described methodology of performing neonatal hypoxia-ischemia treatment in mouse pups. The authors include surgical techniques, electrode implantation as well as examples of analyzed data. For a reader to be able to reproduce methodology described in this paper, more details would be necessary around how electrodes are placed in the animal. Methodology about data acquisition and analysis is insufficiently described in the manuscript.
Major Concerns:
Data acquisition parameters should be included, as well as how data was processed to generate figures. Additionally, several methods such as BSS scoring are cited, but not described directly in the manuscript. In my opinion, it would be useful for the reader to include the details of these techniques directly in this manuscript. Finally, it would be useful to include a very specific and detailed example of what the authors considered to be a seizure. Was there a difference in EEG between convulsive and non-convulsive events?

Thank you for these comments. As you recommended, we have now included more specifics in the protocol regarding protocol to generate power spectrogram figures and BSS, reference lines 310-311, 314-317. We have also included more details on the definition of seizure that we cited in the protocol, lines 305-307.

Specifics:
Figure 1: It is not clear what authors consider to be "seizure" activity. Panel A shows raw traces of what appears to be background activity, isoelectric EEG and some spiking activity. How is seizure defined? Is it possible to show a trace of what is considered to be a "seizure"? How long are events that are considered to be seizures? It would also be useful to include longer raw EEG traces in addition to spectrogram. Generation of spectrogram is not described in the methods.

We have now defined the criteria we used for seizures in this study (based on previous citations) in the manuscript, lines 305-307. Figure 2 shows a trace of a seizure, with time scale to indicate length of this example seizure. We have also included in the revised manuscript the protocol used to generate the spectrogram, lines 314-317.

Figure 2: The event in B is difficult to interprete. Consider temporally expanding the trace, so that activity is not merged together.

Because of the length of the event used in this example, an expanded trace for the entire event would be cumbersome in a figure. We have demonstrated key patterns on expanded EEG to the right of the trace. Each of these expanded sections represents the correlative region of EEG in the shaded box on the left. The figure legend for this figure has been modified to improve clarity (line 453).

Figure 3: How is data analyzed? Is it percent or raw power? RMS power? Power in certain frequency bands? More details are necessary for reader to be able to replicate this analysis.

We have included more details in the protocol regarding the analysis of EEG data, lines 314-317. Figure 3B is total power as a percentage of each animal’s baseline, see figure 3 legend for these details. Frequency range used is 0-60Hz.
Minor Concerns:
Line 80: What is iBond? Why use it before electrode implantation

iBond is an adhesive which helps fix the electrodes in place. We have added this to the revised protocol, line 92.

Line 88: What happens to electrode after it is placed with acrylic? Is it routed outside the animal somehow? How is it protected from the mother? Does the electrode have some kind of connector? More details in this part would be useful.

The electrode in fixed in place with a low profile headset device. The electrode extensions are connected via flexible cable at the time of EEG recording. We have included these details in the protocol, lines 99-102, 122-123.

Line 88: Consider using "lowered into the brain" or "inserted into the brain" instead of "dropped into the brain"

Thank you, we made this modification, line 100.

Line 95: What temperature are pups maintained at? Is humidity maintained?

Intermittent body temperature checks are performed using an infrared detector to ensure consistent temperatures between 36-37.5C. The surgical field and hypoxia/recording chamber are on circulating water warming mats. Humidity is the same as the ambient air, not continuously monitored.

Line 98: What are the data acquisition parameters? Sampling rate? Instructions about how to make the "custom op-amp" cables would be useful for reproducibility

We have included additional detail on the data acquisition parameters, please see lines 124-125. We digitize the data at 1000Hz with a 1K gain using the grass amplifier. EEG signal is reviewed using LabChartPro (v8) using a band pass filter between 3 and 70Hz. Details on custom op-amp cables are now included on lines 62-72.

Line 106: What are the gain and other settings on the amplifier. More details would be useful

We have included additional details in the protocol, lines 124-125. We digitize the data at 1000Hz with a 1K gain using the grass amplifier.

Line 108: Consider using "Disconnecting" instead of "unhooking"

Thank you, we made this modification, line 126.

Line 119: Which carotid is being ligated? Right or Left? How is the vagus nerve separated from the artery? Is 1 knot enough to completely occlude the blood flow? With one knot, is there reperfusion in the days following the procedure?

The left carotid is ligated, see line 135. A dissecting microscope is used to visualize the anatomy. The vagus nerve is identified, gently separated from the artery using blunt dissection, lines 137-139. Ligation is accomplished using a double knot, as previously published by several groups (Rice 1981, Burnsed 2015, Ferriero 2019). While reperfusion may occur days to weeks after the procedure, this does not affect the results of this experiment, which examines only acute seizures in this injury.

Line 126: How long is recovery time between carotid ligation and hypoxia?

Recovery time is 1 hour, this is included in the protocol, see line 146.

Line 128: Define FiO2 abbreviation

Thank you, we made this modification, line 148.

Line 135: Consider using "Disconnected" instead of "unhooked"

Thank you, we made this modification, line 301 .

Line 166: Consider using "amplitude was reduced" instead of "amplitude on EEG declined"

Thank you, we made this modification, line 342.