Dual-modal metabolic analysis reveals hypothermia-reversible uncoupling of oxidative phosphorylation in neonatal brain hypoxia-ischemia

Reviewer #1 (Public review):

Summary:

This manuscript addresses an important problem of the uncoupling of oxidative phosphorylation due to hypoxia-ischemia injury of the neonatal brain and provides insight into the neuroprotective mechanisms of hypothermia treatment.

Strengths:

The authors used a combination of in vivo imaging of awake P10 mice and experiments on isolated mitochondria to assess various key parameters of the brain metabolism during hypoxia-ischemia with and without hypothermia treatment. This unique approach resulted in a comprehensive data set that provides solid evidence for the derived conclusions.

Weaknesses:

(1) The experiments were performed acutely on the same day when the surgery was performed. There is a possibility that the physiology of mice at the time of imaging was still affected by the previously applied anesthesia. This is particularly of concern since the duration of anesthesia was relatively long. Is it possible that the observed relatively low baseline OEF (~20%) and trends of increased OEF and CBF over several hours after the imaging start were partially due to slow recovery from prolonged anesthesia? The potential effects of long exposure to anesthesia before imaging experiments were not discussed.

(2) The Methods Section does not provide information about drugs administered to reduce the pain. If pain was not managed, mice could be experiencing significant pain during experiments in the awake state after the surgery. Since the imaging sessions were long (my impression based on information from the manuscript is that imaging sessions were ~4 hours long or even longer), the level of pain was also likely to change during the experiments. It was not discussed how significant and potentially evolving pain during imaging sessions could have affected the measurements (e.g., blood flow and CMRO2). If mice received pain management during experiments, then it was not discussed if there are known effects of used drugs on CBF, CMRO2, and lesion size after 24 hr.

(3) Animals were imaged in the awake state, but they were not previously trained for the imaging procedure with head restraint. Did animals receive any drugs to reduce stress? Our experience with well-trained young-adult as well as old mice is that they can typically endure 2 and sometimes up to 3 hours of head-restrained awake imaging with intermittent breaks for receiving the rewards before showing signs of anxiety. We do not have experience with imaging P10 mice in the awake state. Is it possible that P10 mice were significantly stressed during imaging and that their stress level changed during the imaging session? This concern about the potential effects of stress on the various measured parameters was not discussed.

(4) The temperature of the skull was measured during the hypothermia experiment by lowering the water temperature in the water bath above the animal's head. Considering high metabolism and blood flow in the cortex, it could be challenging to predict cortical temperature based on the skull temperature, particularly in the deeper part of the cortex.

(5) The map of estimated CMRO2 (Fig. 4B) looks very heterogeneous across the brain surface. Is it a coincidence that the highest CMRO2 is observed within the central part of the field of view? Is there previous evidence that CMRO2 in these parts of the mouse cortex could vary a few folds over a 1-2 mm distance?

(6) The justification for using P10 mice in the experiments has not been well presented in the manuscript.

(7) It was not discussed how the observations made in this manuscript could be affected by the potential discrepancy between the developmental stages of P10 mice and human babies regarding cellular metabolism and neurovascular coupling

Reviewer #2 (Public review):

Summary:

In this study, authors have hypothesized that mitochondrial injury in HIE is caused by OXPHOS-uncoupling, which is the cause of secondary energy failure in HI. In addition, therapeutic hypothermia rescues secondary energy failure. The methodologies used are state-of-the art and include PAM technique in live animal , bioenergetic studies in the isolated mitochondria, and others.

Strengths:

The study is comprehensive and impressive. The article is well written and statistical analyses are appropriate.

Weaknesses:

(1) The manuscript does not discuss the limitation of this animal model study in view of the clinical scenario of neonatal hypoxia-ischemia.

(2) I see many studies on Pubmed on bioenergetics and HI. Hence, it is unclear what is novel and what is known.

(3) What are the limitations of ex-vivo mitochondrial studies?

(4) PAM technique limits the resolution of the image beyond 500-750 micron depth. Assessing basal ganglia may not be possible with this approach.

(5) Hypothermia in present study reduces the brain temperature from 37 to 29-32 degree centigrade. In clinical set up, head temp is reduced to 33-34.5 in neonatal hypoxia ischemia. Hence a drop in temperature to 29 degrees is much lower relative to the clinical practice. How the present study with greater drop in head temperature can be interpreted for understanding the pathophysiology of therapeutic hypothermia in neonatal HIE. Moreover, in HIE model using higher temperature of 37 and dropping to 29 seems to be much different than the clinical scenario. Please discuss.

(6) NMR was assessed ex-vivo. How does it relate to in vivo assessment. Infants admitted in Neonatal intensive Care Unit, frequently get MRI with spectroscopy. How do the MRS findings in human newborns with HIE correlate with the ex-vivo evaluation of metabolites.

Reviewer #3 (Public review):

Sun et al. present a comprehensive study using a novel photoacoustic microscopy setup and mitochondrial analysis to investigate the impact of hypoxia-ischemia (HI) on brain metabolism and the protective role of therapeutic hypothermia. The authors elegantly demonstrate three connected findings: (1) HI initially suppresses brain metabolism, (2) subsequently triggers a metabolic surge linked to oxidative phosphorylation uncoupling and brain damage, and (3) therapeutic hypothermia mitigates HI-induced damage by blocking this surge and reducing mitochondrial stress.

The study's design and execution are great, with a clear presentation of results and methods. Data is nicely presented, and methodological details are thorough.

However, a minor concern is the extensive use of abbreviations, which can hinder readability. As all the abbreviations are introduced in the text, their overuse may render the text hard to read to non-specialist audiences. Additionally, sharing the custom Matlab and other software scripts online, particularly those used for blood vessel segmentation, would be a valuable resource for the scientific community. In addition, while the study focuses on the short-term effects of HI, exploring the long-term consequences and definitively elucidating HI's impact on mitochondria would further strengthen the manuscript's impact.

Despite these minor points, this manuscript is very interesting.