3D Doppler Ultrasound Imaging of Cerebral Blood Flow for Assessment of Neonatal Hypoxic-Ischemic Brain Injury in Mice

Guofang Shen, Kayla Sanchez, Shirley Hu, Zhen Zhao, Lubo Zhang, Qingyi Ma


Cerebral blood flow (CBF) acutely reduces in neonatal hypoxic-ischemic encephalopathy (HIE). Clinic studies have reported that severe CBF impairment can predict HIE outcomes in neonates. Herein, the present study uses a non-invasive 3D ultrasound imaging approach to evaluate the changes of CBF after HI insult, and explores the correlation between CBF alterations and HI-induced brain infarct in mouse pups. The neonatal HI brain injury was induced in postnatal day 7 mouse pups using the Rice-Vannucci model. Non-invasive 3D ultrasound imaging was conducted to image CBF changes with multiple frequencies on mouse pups before common carotid artery (CCA) ligation, immediately after ligation, and 0 or 24 hours after HI. Vascularity ratio of the ipsilateral hemisphere was acutely reduced after unilateral ligation of the CCA alone or in combination with hypoxia, and partially restored at 24 hours after HI.


Hypoxic-ischemic encephalopathy (HIE) is a neurologic condition that results from brain injury in neonates due to perinatal conditions such as pre-eclampsia and birth asphyxia. HIE is estimated to occur in 1–8 per 1000 births and is one of the leading causes of morbidity and mortality in infants worldwide [1,2]. At present, there are very limited therapeutic options available besides hypothermia [3]. Alteration in cerebral blood flow (CBF) and perfusion is the predominant factor contributing to brain damage in the development brain, and may be related to the development of HIE [4,5]. Clinic studies have shown that severe CBF impairment can predict HIE outcomes in neonates [6,7].

Material and methods

Animals and surgical procedures

All procedures and protocols were approved by the Institutional Animal Care and Use Committee of Loma Linda University and followed the guidelines by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Pregnant CD1 mice purchased from Charles River Laboratories (Portage, MI, USA) and their offspring were used for this study. Mice were housed under a 12-h light–dark cycle in Loma Linda University Animal Care Facility and had access to food and water ad libitum. A total of 168 pups of both sexes were used in the present study. Pups without brain lesion after HI insult were excluded. Rigor study criteria were followed including randomization, blinding, predefined exclusion and inclusion criteria, etc. Animals were sacrificed using isoflurane inhalation. Post-operative ketoprofen (2mg/kg, IM) was administered as necessary to relieve any signs of pain.


To optimize the parameters for CBF detection, head ultrasound was performed under three chosen pulse repetition frequencies (PRFs) 2, 5 or 8 kHz. A ratio of ipsilateral vascularity to contralateral vascularity (ratioi/c) was calculated to reflect CBF impairment. Head ultrasound imaging was performed before CCA ligation (Normal), immediately after ligation (Ligation), and immediately after hypoxia treatment (HI-0 hour). The result showed that the ipsilateral and contralateral hemispheres had similar levels of CBF under different PRFs (0.97±0.03, 2 kHz; 1.09 ±0.08, 5 kHz; 1.07±0.07, 8 kHz) before CCA ligation (Fig 1A–1D, Normal). Immediately after CCA ligation, a reduction of CBF was observed in the ipsilateral hemisphere (Fig 1A, Ligation), which was further reduced after hypoxia treatment detected immediately (Fig 1A, HI-0 hour).


In the present study, we attempted to use non-invasive color doppler ultrasound to evaluate the brain injury in a neonatal HI mouse model. We demonstrated that the CBF was acutely reduced in the ipsilateral hemisphere of the mouse brain after HI insult, which was somehow recovered, but remains significantly reduced 24 hours after HI insult. Moreover, we identified that CBF alteration was moderately correlated with the brain infarct size induced by neonatal HI insult. We further assessed the neuroprotective effect of CNP administered via intranasal route, and found that intranasal CNP administration protected ipsilateral CBF, reduced brain infarct size, and improved long-term neurological function after HI insult. Our finding suggests that 3D ultrasound imaging of CBF was a useful tool which can be used for evaluation of neonatal HI brain injury in mouse model.


In conclusion, our study shows that 3D ultrasound is an effective non-invasive method for monitoring acute CBF changes and brain injury in mouse pups after HIE. Using this approach, we demonstrate that intranasal CNP improves brain perfusion and protects the neonatal brain from HI brain injury.


We are grateful to Jiali Wang at Kaiser Permanente Southern California Permanente Medical Group for his assistance in data analysis.

Citation: Shen G, Sanchez K, Hu S, Zhao Z, Zhang L, Ma Q (2023) 3D doppler ultrasound imaging of cerebral blood flow for assessment of neonatal hypoxic-ischemic brain injury in mice. PLoS ONE 18(5): e0285434.

Editor: Masaki Mogi, Ehime University Graduate School of Medicine, JAPAN

Received: February 8, 2023; Accepted: April 21, 2023; Published: May 9, 2023

Copyright: © 2023 Shen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript.

Funding: This work was supported by the National Institutes of Health grant NS112404 and NS126583-01A1 to Dr Ma. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

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