Dual-modal metabolic analysis reveals hypothermia-reversible uncoupling of oxidative phosphorylation in neonatal brain hypoxia-ischemia
Department of Biomedical Engineering, Washington University in St. Louis, United States
Department of Biomedical Engineering, University of Virginia, United States
Institute of BioPharmaceutical Sciences, National Sun Yat-Sen University, Taiwan
Department of Neuroscience, University of Virginia, United States
Center for Brain Immunology and Glia (BIG), University of Virginia, United States
Department of Life Sciences and Institute of Genome Sciences, National Yang Ming Chiao Tung University, Taiwan
Department of Radiology, Cincinnati Children’s Hospital Medical Center, United States
Department of Anesthesiology, Taipei Veterans General Hospital, Taiwan
Department of Anesthesiology, College of Medicine, National Yang Ming Chiao Tung University, Taiwan
Fetal-Neonatal Neuroimaging and Developmental Science Center, Boston Children’s Hospital, United States
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Dual-modal metabolic analysis reveals hypothermia-reversible uncoupling of oxidative phosphorylation in neonatal brain hypoxia-ischemia
study that utilized in vivo optical measurements of the cortical metabolic rate of O2 and blood flow, as well as measurements in isolated mitochondria to assess the uncoupling of the oxidative phosphorylation due to hypoxia-ischemia injury of the neonatal brain, and effects of the hypothermia treatment. The combination of state-of-the-art optical measurements, mitochondrial assays, and the use of various control experiments provides
evidence for the derived conclusions. This work will be of interest to those in the mitochrondrial metabolomics, brain injury and hypoxia fields.
https://doi.org/10.7554/eLife.100129.3.sa0
: Findings that have theoretical or practical implications beyond a single subfield
: Appropriate and validated methodology in line with current state-of-the-art
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Hypoxia-ischemia (HI), which disrupts the oxygen supply-demand balance in the brain by impairing blood oxygen supply and the cerebral metabolic rate of oxygen (CMRO
), is a leading cause of neonatal brain injury. However, it is unclear how post-HI hypothermia helps to restore the balance, as cooling reduces CMRO
. Also, how transient HI leads to secondary energy failure (SEF) in neonatal brains remains elusive. Using photoacoustic microscopy, we examined the effects of HI on CMRO
in awake 10-day-old mice, supplemented by bioenergetic analysis of purified cortical mitochondria. Our results show that while HI suppresses ipsilateral CMRO
-surge post-HI, associated with increased mitochondrial oxygen consumption, superoxide emission, and reduced mitochondrial membrane potential necessary for ATP synthesis—indicating oxidative phosphorylation (OXPHOS) uncoupling. Post-HI hypothermia prevents the CMRO
-surge by constraining oxygen extraction fraction, reduces mitochondrial oxidative stress, and maintains ATP and N-acetylaspartate levels, resulting in attenuated infarction at 24 hr post-HI. Our findings suggest that OXPHOS-uncoupling induced by the post-HI CMRO
-surge underlies SEF and blocking the surge is a key mechanism of hypothermia protection. Also, our study highlights the potential of optical CMRO
measurements for detecting neonatal HI brain injury and guiding the titration of therapeutic hypothermia at the bedside.
The human brain constitutes only 2% of the body mass, but utilizes ~20% of body’s oxygen consumption (
). Disruptions in cerebral blood flow and oxygen supply cause a spectrum of brain injuries, including adult ischemic stroke and neonatal hypoxic-ischemic encephalopathy (HIE;
). In adult ischemic stroke, a >60% reduction in the cerebral metabolic rate of oxygen (CMRO
). However, the impacts of HIE on cerebral oxygen metabolism are far less certain due to the challenge of measuring CMRO
in infant brains by magnetic resonance imaging (MRI) or positron emission tomography (PET). Studies in animal models of HIE have revealed an initial recovery of brain adenosine triphosphate (ATP) levels during a short latent period after hypoxia-ischemia (HI), followed by a precipitous decline known as the secondary energy failure (SEF) that signifies looming infarction (
). However, the mechanisms of post-HI SEF and how therapeutic hypothermia protects neonatal brains against HI injury remain uncertain (
measurements with recently developed bedside optical instruments, free of MRI’s susceptibility to infant motion and PET’s radiotoxicity, can detect HIE brain injury in a manner similar to adult ischemic stroke (
Mitochondrial bioenergetics is vital to cellular functions and survival, and dysregulated oxidative-phosphorylation (OXPHOS) may promote neonatal HI brain injury (
). While the phosphorylation efficiency of mitochondrial respiration (P/O ratio) remains stable over a wide range of substrate concentrations, it declines under hypoxia through a process known as OXPHOS-uncoupling (
), which is indicated by the rise of oxygen consumption despite a reduction of the mitochondrial membrane potential that is needed for ATP synthesis (
). Although OXPHOS-uncoupling has been implicated in decoupling the contractive strength from oxidative metabolism after cardiac ischemia (
), its role in HIE is unknown. We hypothesized that OXPHOS-uncoupling may be associated with mitochondrial injury in HIE, serving as the cause of post-HI SEF and a target for therapeutic hypothermia.
To test this hypothesis, we applied a head-restrained photoacoustic microscopy (PAM) technique to measure CMRO
during and immediately after HI in awake 10-day-old (P10) mice. Using multi-parametric PAM, we simultaneously acquired high-resolution images of cerebral blood flow (CBF), the oxygen saturation of hemoglobin (sO
), and the total concentration of hemoglobin (C
), which can be combined to calculate CMRO
and see Methods for details of the PAM system). The use of awake mouse neonates avoided the confounding effects of anesthesia on CBF and CMRO
). In addition, we measured the oxygen consumption rate (OCR), reactive oxygen species (ROS), and the membrane potential of mitochondria that were immediately purified from the same cortical area imaged by PAM. This dual-modal analysis enabled a direct comparison of cerebral oxygen metabolism and cortical mitochondrial respiration in the same animal. Moreover, we compared the effects of therapeutic hypothermia on oxygen metabolism and mitochondrial respiration and correlated the extent of CMRO
reduction with the severity of infarction at 24 hr after HI. Our results suggest that blocking HI-induced OXPHOS-uncoupling is an acute effect of hypothermia and that optical detection of CMRO
Multi-parametric photoacoustic microscopy (PAM) of hemodynamic and oxygen-metabolic responses of uninjured neonatal mouse brains to hypothermia or hypoxia.
) Schematic of the head-restrained multi-parametric PAM system. PA, photoacoustic; PD, photodiode; HWP, half-wave plate; EOM, electro-optical modulator; PBS, polarizing beam splitter; NDF, neutral-density filter; PM-SMF, polarization-maintaining single-mode fiber; BPF, band-pass filter; DBS, dichroic beam splitter; BS, beam sampler; SMF, single-mode fiber; DL, doublet; CL, correction lens; UT, ring-shaped ultrasonic transducer; WT, water tank. (
) Photograph of the placement of an awake 10-day-old (P10) mouse wearing the head plate in the PAM system. Note that the water tank is removed to better show the mouse and related parts for head-restrained awake-brain imaging. (
) Illustration of the imaging fiel