Dysregulated hippocampal fatty acid metabolism following intermittent hypoxemia-induced neonatal brain injury is rescued by treatment with acetate

Domain: biology
Published: 2025-12-14
Editorial outcome: minor_revision

Abstract
The brain is a lipid-rich organ that experiences rapid growth and development after birth in a period hallmarked by extensive lipid synthesis. We still lack a fundamental understanding of lipid metabolism during this critical time of brain development and how these dynamics occur in infants born extremely preterm (<28 weeks of gestation) suffering from brain injuries. Using an established model of neonatal brain injury due to intermittent hypoxemia, we recapitulate hippocampal-dependent cognitive impairments and examine the extent of changes in the brain’s lipid profile. Our results show changes in hippocampal lipid composition and abnormal fatty acid profile. Furthermore, we provide evidence of an increase in mitochondrial fatty acid β-oxidation, a process that is not classically thought of occurring in the developing brain. We find that a specific alternative fuel, acetate, spares fatty acids from mitochondrial β-oxidation. Here, we show that treatment with acetate in vivo in the form of glycerol-triacetate promotes functional recovery and restores hippocampal fatty acid profile after neonatal brain injury.

Introduction
The developing brain undergoes rapid and complex changes, creating the foundation for a life of cognitive processing. During the third trimester and first year of life, large quantities of fatty acids are required as building blocks to make complex lipids, which in turn, build a lipid-rich brain architecture1,2. Thus, this period is characterized by both significant uptake and de novo synthesis of fatty acids in the brain. In contrast, mitochondrial β-oxidation, the other major fate of fatty acids, is negligible, especially compared to the adult brain3,4. It remains unknown whether these processes – fatty acid synthesis and oxidation – are dysregulated in neonates born extremely preterm (EPT; <28 weeks of gestation).

Neonatal brain injury is a major contributor to long-term neurodevelopmental delays5. It is well documented that nearly all infants born EPT have episodic intermittent hypoxemia (apnea of prematurity)6–9. These brief, repeated episodes (50–100 per day) of apnea lead to low oxygen saturation and are often addressed in the neonatal intensive care unit by providing supplemental O2 to restore oxygen levels. Unfortunately, these episodes of intermittent hypoxia continue during the entire period until these infants achieve corrected full-term age7–9. Clinical observational studies suggest that the frequency, severity, and duration of these episodes impact neurodevelopmental outcomes8,10. However, cognitive impacts are often not evident until school age when children confront more complex social and behavioral interactions. Indeed, studies of children born EPT showed impaired executive and cognitive deficits as well as structural changes to the brain (such as lower hippocampal volume)7–9,11,12. Both human and animal studies have shown that the hippocampus is vulnerable to injury and hippocampal volume is correlated with memory impairments at childhood10,12,13.

To study the effects of intermittent hypoxemia, we used a clinically relevant mouse model of neonatal brain injury from apnea of prematurity14,15. This model of intermittent hypoxia (IHx) recapitulates the repeated hypoxic episodes from apnea of prematurity observed in human infants born EPT that result in neuroanatomical and neurofunctional deficits15. The exact cellular and biochemical mechanisms responsible for impaired memory and learning in children born EPT remain unknown. The timing of preterm brain injury, which coincides with significant uptake and de novo fatty acid synthesis and accretion, suggest that fatty acid metabolism in the developing brain may be implicated. We hypothesize that IHx disrupts hippocampal development by dysregulating fatty acid metabolism. Therefore, our study aimed to delineate the effect of IHx on fatty acid metabolism during the neonatal period.

In this study, using our mouse model of IHx, we recapitulated the long-term hippocampal-dependent cognitive deficits observed in children born EPT and discovered changes in brain lipid metabolism. We report that IHx led to alterations in hippocampal lipid and fatty acid profiles along with increased fatty acid oxidation in astrocytes. This fatty acid oxidation occurred acutely and, interestingly, persisted for several days during the recovery period in normoxia conditions. Fatty acid oxidation was significantly reduced upon exposure to the alternative fuel, acetate. Importantly, in vivo intervention using oral administration of glycerol-triacetate, a clinically feasible source of acetate, rescued hippocampal fatty acids from oxidation, recovered total lipid fatty acid profile, and resulted in improved cognitive deficits.

Results
Injury from intermittent hypoxia alters hippocampal lipid composition
It is established that the first several postnatal days in rodent (approximately postnatal days 0–10) brain development is analogous to the third trimester in human fetal brain development16,17. Mice underwent intermittent hypoxemia (IHx) during postnatal days (P) 1–11 and were compared to normoxia controls (Nx) (Fig. 1a). Similar to children born EPT, mice that underwent neonatal IHx (P1-P11) had significantly poor performance on recognition and spatial memory tasks at postnatal day 30 (P30) in both males and females compared to Nx controls (Fig. 1b, c, Supplementary Fig. 1a, b). At P30, the body and brain weight of IHx-injured mice was lower compared to age-matched Nx controls, reflecting the effects of preterm birth on optimal growth in children born EPT (Supplementary Fig. 1c, d)18–21.

The exact cellular and molecular mechanisms of preterm brain injury are still unknown. EPT and apnea of prematurity occur at a time of extensive lipid synthesis that is concurrent with rapid structural brain growth and development1,2. Therefore, we studied brain lipid metabolism, specifically within the hippocampus, in our model of IHx. Within the hippocampus, the percent of total lipids per mg of tissue was not changed at P30 (Fig. 1d, Supplementary Fig. 1e). However, hippocampal lipid profile at this age showed that IHx during the neonatal period altered lipid composition (Fig. 1e–h, Supplementary Table 1). Unsupervised principal component analysis (PCA) showed minimal overlap between groups (Fig. 1e). Subsequent supervised classification by partial least-squares discriminant analysis (PLS-DA), hierarchical clustering, and volcano plot revealed significant differences in lipid classes (Fig. 1f–h, Supplementary Table 1). Specifically, our results show that at P30, 112 lipids were significantly changed in mice that underwent IHx compared to Nx (Fig. 1f–h, Supplementary Table 1).

The majority of lipids in the brain are synthesized locally via de novo lipogenesis, a process that occurs at high rates during the early neonatal period and declines with age22–24. To determine whether de novo lipogenesis was affected by IHx during development, we measured acetate incorporation into hippocampal lipids at several time points (P11, P17, P22, and P30). The results showed that de novo lipogenesis was comparable between Nx and IHx at all ages in both males and females (Supplementary Fig. 1f). Lipids require fatty acids as building blocks, therefore, we explored if there were any changes in total lipid fatty acid composition following IHx at P30 (Fig. 2, Supplementary Tables 2, 3)2,24–26. Fatty acid class analysis showed that while saturated fatty acids were comparable between Nx and IHx, the profile significantly differed (Fig. 2c, d). Specifically, the percent of saturated fatty acids composed of a ≥20-carbon chain length were decreased following IHx in males and females while C16:0 (palmitate), one of the most abundant saturated fatty acids (~24%), was increased in males only (Fig. 2a, b, e, f, Supplementary Table 2 and 3). The percent of total unsaturated ω−9 fatty acids was significantly decreased after IHx in males only (Fig. 2c, d). Notably, C18:1(n−9) (oleic acid) which constitutes ~15% of total fatty acids, was one of the ω−9 species affected in males (Fig. 2a, Supplementary Table 2). Furthermore, within the ω−9 fatty acid class, the percent of ≥20-carbon long fatty acids were trending towards or significantly decreased in IHx-injured hippocampus in both sexes (Fig. 2e, f). In contrast to the reduced levels of these long and very long-chain fatty acids (VLCFA), the levels of arachidonic acid (C20:4n-6), the essential fatty acid belonging to the ω-6 fatty acid class, was increased in males while no differences were observed in females (Fig. 2a, b, Supplementary Tables 2, 3). Together, these changes in hippocampal lipid composition at P30 in the IHx-injured group reflect prolonged dysregulations in lipid homeostasis during the recovery phase from neonatal intermittent hypoxia.

Next, we investigated whether changes in lipid composition began immediately after the injury and whether they resembled those observed at P30. Hippocampal lipid composition from IHx-injured mice at P11 displayed less changes compared to P30 (Fig. 3a–e, Supplementary Table 4). PCA showed some overlap between P11 Nx and IHx groups (Fig. 3a). Furthermore, PLS-DA revealed that the Nx group was completely separated from the IHx group indicating significant differences in their lipid profile (Fig. 3b). Using hierarchical clustering and volcano plot analysis, we identified the lipid species contributing to the separation of the groups and the direction of their change (Fig. 3c, d, Supplementary Table 4). In contrast to P30, only forty-seven lipids were identified as significantly changed. Phosphatidylserine (PS), the most abundant anionic phospholipid found in brain lipid membranes, was markedly decreased by IHx at P11 and P30 (Figs. 1g, h, 3c–e, Supplementary Tables 1 and 4)27. The sum of ceramides was decreased after IHx at P11 but subsequently recovered and exceeded those in the Nx group by P30 (Figs. 1h, and 3c–e, Supplementary Tables 1, 4). We then performed total lipid fatty analysis in the hippocampus at P11 (Fig. 3f–i, Supplementary Fig. 1g, h). In contrast to data at P30, hippocampal levels of long-chain fatty acids at P11 showed minimal changes between Nx and IHx groups (Supplementary Fig. 1g, h and Supplementary Tables 5, 6). Specifically, at P30 we observed significant differences in the levels of palmitate (C16:0) and oleic acid (18:1n−9), while at P11 the levels of these fatty acids were comparable between Nx and IHx in both males and females (Fig. 2a, b, Supplementary Fig. 1g, h, Supplementary Tables 2, 3, 5, 6). Similarly, while the percent of ≥20 carbon-long saturated and unsaturated ω−9 fatty acids were decreased at P30 in the IHx group, no such differences were detected at P11 between Nx and IHx groups (Figs. 2e, f and 3h, i).

Next, we evaluated whether the data from murine experiments recapitulate human total lipid fatty acid profile of term human hippocampus (Supplementary Fig. 2). We compared human hippocampal total lipid fatty acid profile to data obtained from Nx controls at P11 and P30 using post-mortem hippocampi from term newborns (Supplementary Fig. 2a–d). This comparison showed compelling similarities to Nx controls at P11 but not at P30 (Supplementary Fig. 2c, d). The rapid brain growth and development is characterized by extensive local synthesis of ≥20 carbon-long saturated fatty acids, which were increased between P11 and P30 Nx mice (P11-~0.5% vs P30-~2.4%) (Supplementary Fig. 2d). Next, we examined the hippocampal total lipid fatty acid profile between full term (38–40 weeks of gestation) at ~1.6-month-old average postnatal age and extremely preterm infants (≤28 weeks of gestation) at corrected similar age (Supplementary Fig. 2a, e–g, Supplementary Table 7). These results showed that the fatty acid composition of the hippocampus in extremely preterm infants did not differ from those born full term (Supplementary Fig. 2e–g). Moreover, our findings provide evidence that P11 murine hippocampal fatty acid profile correlates to human hippocampus in early infancy (1–3 months) and that the fatty acid results from the preterm compared to term infants are similar to the results from the IHx murine experiments at P11. Taken together, our data indicate that changes in brain lipid composition begin to emerge in IHx-injured mice as early as P11 but become prominent at a later age.

Since the brain depends on a continuous supply of substrates for metabolism and de novo lipid synthesis, we first sought to determine whether the observed dysregulated lipid metabolism is due to an altered supply of nutrients following IHx. The developing brain relies on glucose extensively for local de novo synthesis of fatty acids and as a substrate for energy22,24. In contrast to the adult brain, the developing brain uses alternative substrates such as ketones and lactate for energy22,24. No hypoglycemia was observed in the IHx group (Supplementary Fig. 3a). Using 3H-2-Deoxy-D-glucose (2-DG), we determined that glucose uptake in the hippocampus was comparable between the Nx and IHx males (Supplementary Fig. 3b). Of note, only females at P11 in the IHx group showed lower levels of blood glucose (Supplementary Fig. 3a) and decreased tracer uptake in the hippocampus at P11 and P30 (Supplementary Fig. 3b). Brain glucose 2-DG uptake was not correlated with plasma 2-DG (Supplementary Fig. 3b-d). As expected, systemic lactate levels were increased in the IHx group at P11 only (Supplementary Fig. 3e). Circulating levels of non-esterified fatty acids (NEFA) were significantly decreased in both sexes at P22 in the IHx-treated group, which, interestingly, occurred immediately after weaning (Supplementary Fig. 3f). NEFA metabolism generates ketones and acylcarnitines (ACs), which are intermediates of fatty acid oxidation. Indeed, the plasma levels of the ketone body β-hydroxybutyrate (βHB) and of acylcarnitines (ACs) were increased at P11 and P17 compared to Nx controls (Supplementary Figs. 3g, 4a–d, Supplementary Table 8). To determine whether blood AC levels reflect brain ACs, we measured ACs in the hippocampus and the results showed that total ACs were increased only at P11 indicating that fatty acids were mobilized for subsequent generation of acetyl-CoA (Supplementary Fig. 4e–h, Supplementary Table 9). Furthermore, there was no deficiency of carnitine in the hippocampus of the IHx-injured group (Supplementary Fig. 4e). In fact, the level of free carnitine in the brain was increased, thus being readily available to facilitate fatty acid entry into the mitochondria via the carnitine shuttle for subsequent β-oxidation. Altogether, these data show that dysregulated brain lipid homeostasis is not the result of a limited supply of nutrients.

These substrates provide the mitochondrial generated carbons for subsequent de novo lipid synthesis. Therefore, we question whether changes in mitochondrial content could compromise the brain’s ability to utilize these substrates after the injury. We quantified mitochondrial content by digital PCR and our results revealed that at P11 the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (gDNA) was lower following IHx but recovered by P30 (Fig. 4a, Supplementary Fig. 5a). Since we observed the changes in mitochondrial content immediately after IHx, we performed an unbiased proteomics analysis of the hippocampus to better understand the changes that occur at this age (P11) and to identify proteins and pathways that may be responsible for abnormal lipid composition following IHx.

IHx affects the expression of lipid metabolism genes and proteins
Immediately after IHx (P11), pathway analysis of the proteomics revealed that the most significantly downregulated proteins belong to the metabolism pathway (Fig. 4b, c, Supplementary Fig. 5b–e). Within the metabolism pathway, we found that metabolism of lipids was one of the top three most downregulated pathways (Fig. 4b, Supplementary Fig. 5b, d). Because lipid-related proteins were downregulated in these pathways at P11, we assessed the temporal expression of genes crucial for lipid metabolism, specifically genes involved in fatty acid synthesis and oxidation, lipid regulation, and cholesterol and ketone metabolism in the hippocampus (Fig. 4d, e, Supplementary Fig. 6). The mRNA data showed that IHx resulted in a transient decrease of genes involved in lipid metabolism. Specifically, following IHx, the expression of genes responsible for fatty acid oxidation (Cpt2), synthesis of long-chain fatty acids and cholesterol (Scd2, Elovl6, Hmgcr), and lipid regulators (Acc1) were decreased at P11, P17, and P22 (Fig. 4d, e, Supplementary Fig. 6a–d). However, they not only recovered by P30, but some of these genes were upregulated at this age (Srebp2, Hmgcr) (Supplementary Fig. 6c, d). Similarly, the expression of Fasn – an enzyme system responsible for de novo synthesis of primarily palmitate (C16:0) - was downregulated at P11 and subsequently upregulated at P30 (Fig. 4d and Supplementary Fig. 6a). Elovl5, which is essential for the elongation of ω-3 and ω-6 fatty acids, was not changed (Fig. 4d and Supplementary Fig. 6a). These results showed that IHx downregulates the expression of lipid metabolism related genes as early at P11, however, these changes were transient and recovered by P30. Moreover, it is important to point out that these genes are subject to post-transcriptional, post-translational, and allosteric regulation23,30. While the unbiased proteomics and gene expression analyses highlight interesting findings, they do not assess function. Therefore, in addition to gene expression and proteomics analysis, we then sought to determine the functional alterations by which neonatal intermittent hypoxia results in long-term changes in brain lipid composition.

IHx increases fatty acid oxidation and alters lipid composition at P17
We first tested the dependency of the injured brain on different substrates as fuel for energy production by performing the Seahorse XF Mito Fuel Flex test in single-cell suspensions from the hippocampus (Fig. 5a). We found that the injured brain had a decreased dependency on glucose for fuel at P11 (Fig. 5a). There were no differences observed in mitochondrial glutamine dependency at any of the ages studied (P11, P17, P30) (Fig. 5a). Interestingly, the IHx-injured group had a significant increase in dependency on fatty acids as a fuel at P11, and this persisted up to P17, six days after recovery from IHx (Fig. 5a). At P30, there was a significant decrease in fatty acids dependency in IHx-injured mice compared to Nx (Fig. 5a). To validate our findings, we measured the oxidation of [14C]-substrates to 14CO2 ex-vivo in micro-dissected hippocampi in both groups (Supplementary Fig. 7a). [U-14C]-glucose oxidation was increased only at P11 (Supplementary Fig. 7b) whereas the fatty acid [1-14C]-oleic acid (C18:1) oxidation was increased at P11 and P17 (Fig. 5b, Supplementary Fig. 7c). There were no differences in 14C-acid soluble metabolites (ASM) generated by [1-14C]-oleic acid oxidation (Supplementary Fig. 7d). Because the oxidation of fatty acids remained high during the subsequent 6 days after the injury, we sought to determine whether the developing brain had increased circulating fatty acid uptake into the brain at P17 (Supplementary Fig. 7e–g). Thus, we measured the uptake of 3H-2-bromopalmitate, an analogue of the fatty acid palmitate that cannot be β-oxidized and showed a significant increase in IHx-injured mice (Supplementary Fig. 7e–g). Furthermore, at P17, we measured the oxidation of other alternative substrates. The oxidation of [U-14C]-glutamine, [3-14C]-βHB, and [U-14C]-lactate did not change at P17 in IHx-injured mice compared to Nx controls (Supplementary Fig. 7h–j). Taken together, these data show that immediately after intermittent hypoxia (P11-acute period) and during the recovery period under normoxic conditions (P17 - 6 days after IHx), the developing brain upregulates long-chain fatty acid oxidation to fuel and support oxidative metabolism.

Previous studies showed that neural progenitor cells and astrocytes express the enzymatic machinery for fatty acid oxidation3,4,31. In this study we used a conditional knockout mouse of carnitine palmitoyltransferase-2 (CPT2), an obligatory enzyme for mitochondrial long-chain fatty acid β-oxidation (Supplementary Fig. 7k)3. We determined that [1-14C]-oleic acid oxidation was significantly decreased after IHx in both pan-brain (Nestin-Cre; CPT2B−/−) and astrocyte-specific (Gfap-Cre; CPT2G−/−) knockout mice compared to IHx-injured wild-type mice (Fig. 5c, Supplementary Fig. 7l, m). These results indicate that after IHx, astrocytes are the primary brain cells that oxidize fatty acids.

Since long-chain fatty acids were being extensively oxidized during the recovery from IHx up to P17, we analyzed the fatty acid profile of P17 hippocampus to determine if the fatty acid composition was also affected at this time point. Compared to Nx control, P17 IHx-injured mice displayed changes in total fatty acid composition similar to P30 IHx-injured mice (Figs. 2a, b, 5d, e, Supplementary Tables 2, 3, 10, 11). Notably, IHx had a significant, or trending, lower percent of total brain ω−9 fatty acids in both sexes (Fig. 5f, g). These ω−9 fatty acids are synthesized extensively in the brain and accumulate during development. Specifically, our analysis revealed a reduction in saturated and ω−9 fatty acids composed of ≥20-carbons in IHx-injured mice of both sexes compared to Nx (Fig. 5h, i). These long and VLCFA, are exclusively synthesized de novo in the brain32. In addition, oleic acid (C18:1n−9) was decreased in the IHx-injured mice (male, p=0.082, female p<0.05) (Fig. 5d, e, Supplementary Tables 10, 11). In contrast to decreased levels of ≥20 carbon-long fatty acids, IHx resulted in significant, or trending, increases in the percent of C14:0, C16:1(n-7), 16:1(n-9), C18:3(n-6), and C20:3(n-7) fatty acids in both male and female pups (Fig. 5d, e, Supplementary Tables 10, 11). Furthermore, in males we observed increased levels of the medium-chain fatty acids (C10:0, C12:0, C10:1) as well as C18:2(n−6) compared to Nx controls (Fig. 5d, Supplementary Table 10). Altogether, these data suggest that IHx leads to reduced levels of saturated and unsaturated ω−9 ≥20 carbon-long fatty acids in the hippocampus as early as P17, resulting in a shift in total fatty acid composition.

Next, we aimed to further characterize in situ these lipid fatty acid profile changes following IHx at P17. Using non-targeted matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), we identified several ions corresponding to lipid species containing medium or long-chain saturated and monounsaturated fatty acids that increased after IHx across multiple brain regions (PC(30:0) – m/z 744.4929 [M+K]+ and PC(32:1) – m/z 770.5080 [M+K]+) (Fig. 6a). Conversely, ions corresponding to lipids containing ≥18 carbons saturated and monounsaturated fatty acids were decreased after IHx (PC(36:1) – m/z 826.5701 [M+K]+ and SM(36:1;O2) – m/z 769.5609 [M+K]+) (Fig. 6b). Quantification of ion intensities in the hippocampus showed statistically significant changes in these lipid species (Fig. 6c, d). Furthermore, lipid mapping using MALDI-MSI also enabled us to detect subregional differences. For example, PC(32:1) is highly enriched in the dentate gyrus (white arrows) while SM(36:1;O2) in the CA3 (pink arrow) (Fig. 6a, b). These results corroborate the total lipid fatty acid profile at P17 (Fig. 5d) and provide additional evidence of changes observed in the lipidomic profile at P30 (Fig. 1e–h). Together, these data demonstrate that IHx results in hippocampal region-specific lipid changes and that these lipid changes are congruent with the lipid fatty acid chain composition.

To get a more detailed understanding of the specific lipid changes that occur at P17, six days after recovery from IHx, we performed lipidomic analysis (Fig. 6e–h, Supplementary Table 12). PCA showed a partial overlap between the Nx and IHx groups, whereas the PLS-DA demonstrated distinct separation suggesting that the lipid profile vary between groups (Fig. 6e, f). Indeed, by P17 there were more lipid changes observed compared to the IHx P11 group (47 lipids at P11 vs 76 lipids at P17). Among the top 50 most differentially changed lipids, several phosphatidylethanolamine (PE) lipid species containing 18–20 carbon-long saturated and monounsaturated fatty acids were decreased after IHx (Fig. 6g, Supplementary Table 12). The majority of the lipids that were increased belonged to the sphingolipids lipid class, which included hexoceramides (Hex1Cer), dihexoceramides (Hex2Cer), sphingomyelin, dihydroceramides (DhCer), and ceramides (Fig. 6g, Supplementary Table 12). Volcano plot analysis revealed that there were many more significantly changed lipids displaying a large increase after IHx (Log2-fold change >15) compared to those that were decreased (14 increased vs 2 decreased) (Fig. 6h). Together these data suggest that IHx affects the developmental lipid remodeling occurring in the hippocampus at P17.

The next critical transition of development is switching from a lipid-rich diet (suckling) to consuming a carbohydrate-rich diet. Hence, we determined the lipid composition of the hippocampus at P22, one day after pups weaning (Supplementary Fig. 8a–d, Supplementary Table 13). While PCA resulted in overlapping groups, PLS-DA clearly differentiated the Nx and IHx groups suggesting that the lipid profile continued to differ at P22 (Supplementary Fig. 8a, b). Hierarchical clustering and volcano plot analysis revealed increase levels of several triacylglyceride species (Supplementary Fig. 8c, d, Supplementary Table 13). Moreover, ceramides were still increased at this time point (Supplementary Fig. 8c, Supplementary Table 13). In addition to analyzing the lipid classes, we measured total lipid fatty acids (Supplementary Fig. 8e-g, Supplementary Table 14, 15). Interestingly, in males, while total saturated fatty acids were comparable between Nx and IHx, the levels of palmitate (C16:0) – the primary product of de novo lipogenesis – was increased in the IHx group (Supplementary Fig. 8e, f, Supplementary Table 14). In females, palmitate levels did not differ between Nx and IHx groups (Supplementary Fig. 8e, Supplementary Table 15). The percent of total saturated and ω−9 fatty acids composed of ≥20-carbons showed a trend towards a decrease only in the IHx-injured female mice compared to Nx (Supplementary Fig. 8g). Altogether, our data show that brain palmitate levels were not compromised despite switching to a carbohydrate-rich diet due to weaning (P21) and subsequent decrease in circulating NEFA (P22) (Supplementary Fig. 3f). These results corroborate our findings that de novo lipogenesis is similar between Nx and IHx groups (Supplementary Fig. 1f).

Acetate as an alternative substrate to reduce fatty acid oxidation
The mitochondrial β-oxidation of fatty acids generates acetyl-CoA which enters the tricarboxylic acid (TCA) cycle and enables the generation of adenosine triphosphate (ATP)33. Acetyl-CoA is a key metabolite that serves as a building block for the synthesis of de novo fatty acids, proteins, and amino acids33. Hence, maintaining the pool of acetyl-CoA is critical for cellular metabolism. We reasoned that supplementing the media with a higher concentration of glucose, a substrate that can be metabolized to yield acetyl-CoA, will spare fatty acids from oxidation in the injured brain. However, high glucose concentrations (25mM) in the media did not affect [1-14C]-oleic acid oxidation compared to 5mM glucose at P17 after IHx (Fig. 7a, Supplementary Fig. 9a). Thus, we next measured the oxidation of fatty acids in the presence of acetate, a substrate that can be directly converted to acetyl-CoA. Media supplementation with 2mM acetate resulted in an 86% and 93% reduction in [1-14C]-oleic acid oxidation at P11 and P17, respectively, compared to IHx-injured hippocampus without acetate supplementation (Fig. 7a, Supplementary Fig. 9a, b). Moreover, [1,2-14C]-acetate oxidation increased after IHx and was not affected by the presence of different oleic acid concentrations (Fig. 7b, Supplementary Fig. 9c). These results indicate that acetate supplants fatty acids for oxidation in the IHx-injured hippocampus.

We next sought to determine whether in vivo acetate administration will spare brain fatty acids from oxidation after IHx, and therefore improve neurologic outcome. Glycerol-triacetate (Triacetin) is an FDA-approved compound that consists of three acetate moieties esterified to a glycerol backbone (Fig. 7c)34. Glycerol-triacetate is readily absorbed in the gut, crosses the blood-brain barrier, and has demonstrated non-toxic effects34. Hence, we chose to administer glycerol-triacetate to supply acetate as an alternative fuel for brain metabolism. Glycerol-triacetate was administered to pups by oral gavage (5mg/g body weight) daily from P11 to P17 (Fig. 7c). In the IHx-injured group, treatment with glycerol-triacetate reduced hippocampal [1-14C]-oleic acid oxidation by 71% at P17 compared to glycerol (vehicle) (Fig. 7d, Supplementary Fig. 9d, e).

To assess whether treatment with glycerol-triacetate can promote neurological recovery after neonatal injury, we performed behavioral testing at P30 following IHx. Using mice treated with glycerol-triacetate (treatment) or glycerol (vehicle) from P11 to P17, we performed the novel object recognition (NOR) test at P30 (Fig. 7e and Supplementary Fig. 9f). IHx-mice treated with glycerol-triacetate showed a significantly increased recognition index in the NOR compared to controls, suggesting an improvement in recognition memory (Fig. 7e and Supplementary Fig. 9f). Together, these data show that treatment with glycerol-triacetate spares fatty acids from mitochondrial β-oxidation and improves neurologic outcome.

Treatment with glycerol-triacetate did not restore body and brain weight in IHx-injured mice by P30 and the percent of total lipids per mg of hippocampus remained the same as the Nx controls (Fig. 7f, g and Supplementary Fig. 9g–i). However, total lipid fatty acid analysis showed that treatment with glycerol-triacetate after IHx restored total lipid fatty acid composition (Fig. 7h-l and Supplementary Fig. 9j–m, Supplementary Tables 16, 17). Specifically, the decrease percent of total ω−9 fatty acids in IHx mice was recovered after glycerol-triacetate treatment in both sexes (Fig. 7i, j). The levels of oleic acid (18:1n−9) that we previously demonstrated to be decreased in the P30 IHx males (Fig. 2a, Supplementary Table 2), were similarly decreased in the IHx-glycerol treated males and restored by glycerol-triacetate treatment (Fig. 7h, Supplementary Table 16). Importantly, glycerol-triacetate treatment prevented the IHx-induced low levels of ≥20-carbon saturated and ω−9 fatty acids (Fig. 7h, k, l). The increase levels of C16:0 in IHx-injured males was reversed to normoxia levels after glycerol-triacetate administration (Fig. 7h, Supplementary Table 16). Together, these data show that treatment with glycerol-triacetate restores the levels of ≥20 carbon-long saturated and ω−9 fatty acids in the hippocampus of IHx-injured mice.

In conclusion, our results reveal that brain fatty acid oxidation, which is negligible in the developing brain, is significantly upregulated following neonatal brain injury from IHx. This shift towards brain fatty acid oxidation leads to decreased levels of ≥20 carbon-long and very-long chain saturated and ω−9 fatty acids, which are synthesized in the brain de novo. Ultimately, this change in fatty acid metabolism disrupts highly orchestrated lipid-rich brain development and maturation and leads to sustained behavioral deficits. An alternative fuel-acetate-can spare fatty acids from oxidation following neonatal brain injury. Treatment with glycerol-triacetate, an FDA-approved compound that provides acetate and can be administered by enteral route, may rescue brain lipid metabolism during a critical period of brain development and prevent cognitive delays associated with intermittent hypoxemia in infants born EPT.

Methods
Animals
Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Johns Hopkins School of Medicine in accordance with the guidelines and regulations of the NIH Guide for the Care and Use of Laboratory Animals (protocol #MO23K317). C57BL/6 wild-type mice (Mus musculus) were purchased from the Jackson Laboratory (Jackson ID #000664); Cpt2lox/lox conditional mice from Dr. Michael J. Wolfgang63 (C57BL/6 genetic background) were bred to Nestin- or Gfap-Cre transgenic mice (Jackson ID #003771; B6.Cg-Tg(Nes-cre)1Kln/J and #024098; B6.Cg-Tg(Gfap-cre)77.6Mvs/2J) to generate pan-brain (Cpt2B−/−) or astrocyte-specific (Cpt2G−/−) knockout mice. Mice were bred in-house and the colonies maintained in the animal facility of Johns Hopkins University School of Medicine in ventilated racks with a 12-hour light/dark cycle in a temperature (20-25°C) and humidity (30–70%) controlled room, and had ad libitum access to water and standard chow (5v75 - PicoLab Verified 75 IF, LabDiet).

Intermittent hypoxia
Postnatal day (P)1 pups were randomized to undergo intermittent hypoxia (IHx) or serve as normoxia (Nx) controls, removed from the dam, and placed in sealed containers (Coy Laboratories) at ambient temperature. IHx pups were exposed to 16 cycles of brief 3 min episodes of hypoxia (8% O2) followed by 5 min of re-oxygenation to 21% FiO2. Nx pups were exposed to 16 cycles of regular air. The pups were returned to their dam for 2h, and the 16 cycles of Nx or IHx were repeated. These 32 cycles in total were repeated daily for 11 days until P11. Pups from different litters and from both sexes were used for each experiment and were assessed at P11, P17, P22, and P30 unless otherwise stated. Data from males and females was reported as combined, male only, and female only. The hippocampus was selected a priori for microdissection and analysis due to its role in learning and memory, and its susceptibility to developmental disruption in children EPT. Blood glucose and lactate levels were determined using blood glucose and lactate meters (Nova Abbott). Blood was collected in 5% 0.5M EDTA, and plasma β-hydroxybutyrate (StanBio; 2440) and non-esterified fatty acids (NEFA) (Wako; 999-35691, 999-34791, 999-34891, 999-35191, 276-76491) were measured as per manufacturer’s instructions.

Glycerol-triacetate treatment
Normoxia and IHx-injured mice were randomized at P11 to receive vehicle (glycerol, Millipore Sigma; G5516) or glycerol-triacetate (Triacetin, Millipore Sigma; 1030001000). Glycerol or glycerol-triacetate was administered by oral gavage (5mg/g body weight) daily from P11 to P17.

Human samples
Human hippocampus was obtained from the National Institutes of Health (NIH) NeuroBioBank at University of Maryland, Baltimore, MD, United States (ID #3243). Donors consisted of ~1.6 months-old (average) infants born at term (38–40 weeks; n=4) and infants born extremely premature (≤28 weeks; n=3) with a corrected average age of ~1-month. Both sexes, male and female, were included in this study. None of the donors had a history of hypoxia ischemia, severe infection, or known genetic diseases. Frozen tissues were stored at –80°C.

Gene expression
Total RNA from microdissected hippocampal tissue (n=8/sex/age/group) was extracted using TRIzol (Life Technologies; 15596018), and further purified using the RNeasy Plus Mini Kit (Qiagen; 74134). RNA was quantified by NanoDrop One spectrophotometer (Thermo Fisher Scientific) and converted to cDNA with the High Capacity cDNA Reverse Transcriptase kit (Applied Biosystems; 4368814). Real-Time PCR was performed using 10ng of cDNA, Sso Advanced Universal SYBR Green Supermix (Bio-Rad; 1725271), and primers for the target genes (Supplementary Table 18), and analyzed using the CFX Opus 96 machine (Bio-Rad). Data was normalized to the average Ct values of the housekeeping genes (18S, Ppia, Ywaz, and Osbp) and expressed as arbitrary units of 2-ΔCt.

To determine the mitochondrial DNA (mtDNA) content relative to nuclear DNA, genomic DNA was isolated from microdissected hippocampus using a QiAmp DNA Mini kit (Qiagen, #51304). EvaGreen-based digital PCR was performed by the Johns Hopkins University School of Medicine Genetic Resources Core Facility (RRID:SCR_018669) using samples as a template and primers to amplify the mitochondrial target genes, cytochrome c oxidase I (Co1) and NADH dehydrogenase 1 (Nd1), and the nuclear target gene H19 (Supplementary Table 18). Biological replicates were tested in duplicate using an 8500 partition nanoplate (Qiagen #250021) in a QIAcuity 8; Firmware version 3.1 instrument (Qiagen) according to the manufacturer’s recommendations for EvaGreen. Each assay was run independently. Samples were run at 10ng/ul concentration for the H19 gene and at a 1:100 dilution for the mitochondrial genes. 5ul of the sample at the appropriate concentration was added to 7.2ul of master mix. 12.2ul of master mix was loaded into the nanoplate, and the instrument delivered 2.6ul of the total to be partitioned across each well. Results were analyzed with the QIAcuity Digital Suite v3.1.0.0 software (Qiagen). Concentrations were adjusted for all dilution levels within the software and are reported as copies/ul in the original 10ng/ul sample tube.

Proteomics
16-plex-TMTpro experiment was performed by the Johns Hopkins Mass Spectrometry and Proteomics Facility. Hippocampal protein was extracted from frozen microdissected hippocampal tissue (total n=16; n=4/sex/group; biological replicates) using RIPA lysis buffer system with protease inhibitors (Santa Cruz; sc-24948). The Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific; 23227) was used to determine protein concentration according to the manufacturer’s protocol. Proteolytic Digestion: Protein samples (50ug each) were diluted to 20ul with 50mM triethylammonium bicarbonate (TEAB), reduced with 5ul 50mM Dithiothreitol in 100mM TEAB at 60°C for 45min followed by alkylating with 100mM Chloroacetamide in 100mM TEAB in the dark for 15min at room temperature. A single pot, solid phase sample preparation protocol (SP3)64 using a mixture of Sera-Mag SpeedBeads (GE Healthcare; 45152105050250 and 65152105050250) was used to remove detergents and other non-protein contaminants. The proteins bound to beads were resuspended in 100ul of 100mM TEAB and digested with 3.4ug of Trypsin/LysC (Pierce) overnight at 37°C. The digested peptides were separated from the magnetic beads using a magnetic tube holder and placed into a new 0.5ml Eppendorf centrifuge tube. Isobaric Mass Tag Labeling: Peptides in each of the 16 digested samples were labeled with a unique TMTpro 16-plex reagent (Thermo Fisher; LOT# WH324722) according to the manufacturer’s instructions. All 16 TMT labeled peptide samples were combined and dried by vacuum centrifugation. Peptide fractionation: The combined TMT-labeled peptides (800ug) were aliquoted, and one aliquot of 300ug was dried prior to off-line fractionation. Peptides were re-constituted in 2ml 10mM TEAB buffer and loaded on a XBridge C18 Guard Column (5µm, 2.1×10mm, Waters) at 250µL/min for 8min prior to fractionation on a XBridge C18 Column (5µm, 2.1×100mm column (Waters) using a 0 to 90% acetonitrile in 10mM TEAB gradient over 85min at 250µL/min on an Agilent 1200 series capillary HPLC with a micro-fraction collector. Eighty-four 250ul fractions were collected and concatenated into 24 fractions according to Wang et al. 2011 and dried65. Mass Spectrometry analysis: Peptides in each of the 24 fractions were analyzed on a nano-LC-Orbitrap-Fusion Lumos-IC in FTFT mode (Thermo Fisher Scientific) interfaced with an EasyLC 1100 series by reversed-phase chromatography using a 2%–90% acetonitrile in 0.1% formic acid gradient over 90min at 300nl/min on a 75µm×150mm ReproSIL-Pur-120-C18-AQ column 3µm, 120Å (Dr. Maisch). Survey scans of precursor ions were acquired from 375 to 1500m/z at 120,000 resolution at 200m/z with automatic gain control (AGC) at 4e5 and a 50ms maximum injection time. Precursor ions were individually isolated in a 3 second cycle time within 0.7m/z by data dependent monitoring and 15s dynamic exclusion, and fragmented using an HCD activation collision energy 36. Fragmentation spectra (MS/MS) were acquired using a 1.25e5 AGC and 86ms maximum injection time (IT) at 50,000 resolution. Data analysis: Fragmentation spectra were processed by Proteome Discoverer v2.4 (PD2.4, Thermo Fisher Scientific) and searched with Mascot v.2.8.0 (Matrix Science, London, UK) against RefSeq 2021_204 mouse database. Search criteria included trypsin enzyme, one missed cleavage, 3ppm precursor mass tolerance, 0.01Da fragment mass tolerance, with TMTpro on N-terminus and carbamidomethylation on C as fixed and TMTpro on K, oxidation on M, deamidation on N or Q as variable modifications. Peptide identifications from the Mascot searches were processed within PD2.4 using Percolator at a 5% False Discovery Rate confidence threshold, based on an auto-concatenated decoy database search. Peptide spectral matches (PSMs) were filtered for Isolation Interference <30%. Relative protein abundances of identified proteins were determined in PD2.4 from the normalized median ratio of TMT reporter ions, having signal to noise ratios >1.5, from all PSMs from the same protein. P-values of mean protein ratios for the biological replicates were calculated using the ANOVA method. Technical variation in ratios from our mass spectrometry analysis is less than 10%66. Pathway enrichment analysis: Pathway analysis of differentially expressed proteins (p-value <0.05) was performed using g:Profiler (https://biit.cs.ut.ee/gprofiler/gost). g:SCS was selected as statistical threshold and p-value <0.05 as the significance threshold. Significant pathways from Reactome were selected and the pathways belonging to the first or second hierarchical levels were plotted67. The proteomics data generated in this study have been deposited to the ProteomeXchange Consortium via the PRIDE68 partner repository with the dataset identifier PXD070491.

Behavior
Novel object recognition (NOR) test and Y-Maze spontaneous alternation test. Novel object recognition test: The test was used to assess hippocampal-dependent recognition memory and performed as previously described in ref. 69. In brief, the open field and objects used were always cleaned with 30% ethanol (vol/vol) between each mouse to minimize olfactory cues. Prior to the testing day, P30 mice were habituated to an opaque rectangular box (27×27×20cm) for 5min. In the familiarization phase, mice were placed inside the box and allowed to explore two identical objects, placed equidistant from each other, for a total of 30s or after 5min had passed. The test was repeated 12h later but one object was replaced with a novel object. Mice were placed inside the box and allowed to explore the objects. The experiment stopped when the mouse explored the objects for a total of 30s. The time it took to reach the criterion of 30s and the total time spent exploring each individual object were recorded and analyzed with the ANY-maze video tracking system (v6.3, Stoelting Co., IL). The recognition index was calculated by dividing the amount of time spent on the novel object by the total exploration time (Time in novel / 30sec). Y-Maze spontaneous alternation test: The test was used to assess spatial working memory as previously described in refs. 69, 70. In brief, P30 mice were placed in the center of a Y-shaped maze and allowed to freely explore the three arms (labeled as A, B, and C) for 8 minutes. The activity and arm choice were recorded with a video camera. The Y-maze was cleaned with 30% ethanol (v/v) between mice. The spontaneous alternation percentage was calculated by dividing the number of alternations (three unique arm entries) by the total number of possible triads minus one (Total alternations/[total entries–2]) × 100. All behavioral tests were performed by the investigator blinded to groups and treatments.

Mito Fuel Flex test
The Mito Fuel Flex test (Agilent) was performed using a Seahorse XFe96 analyzer (Agilent) to measure the dependency of brain cells to oxidize glucose, glutamine, and fatty acids to maintain basal respiration. Single-cell suspensions from microdissected hippocampus were prepared using accutase. The Agilent Seahorse Technical Manual was followed to determine the rate of oxidation of each fuel by measuring the oxygen consumption rate (OCR) in the presence or absence of inhibitors for each of the specific fuel pathways. UK5099 was used as an inhibitor of glucose oxidation, etomoxir as an inhibitor of fatty acid oxidation, and BPTES as an inhibitor of glutamine oxidation. All assays were performed and analyzed in real-time by the manufacturer’s software (Wave 2.6.3). The number of cells plated was analyzed with Biotek Cytation 1 (Agilent), and OCR was normalized to protein concentration in each well. The protein concentration was exported directly to the Agilent Seahorse, and raw data was normalized to account for differences in protein concentration within each well.

Lipidomics
Frozen mouse microdissected hippocampal tissue or human hippocampal tissue (Mouse tissue-total n=48; n=6/age/group, biological replicates; human tissue – total n=7, term/control n=4, preterm n=3; biological replicates) were homogenized with PBS buffer at a concentration 100mg/ml. The total lipid extracts of the brain tissue were prepared using a revised MTBE lipid extraction method71. Briefly, 10ul of brain homogenate, 400ul of ice-cold methanol, and 10ul of internal standard (EquiSPLASH, Avanti Polar Lipids, Alabaster, AL) were combined, vortex mixed for 30s, and then incubated at 4°C for 10min with 650rpm shaking. Next, 500ul of ice-cold MTBE was added, vortex mixed for 30s and incubated at 4°C for 1h with 650rpm shaking. After incubation, 500ul of ice-cold H2O was added, vortex mixed for 30s and incubated at 4°C for 15min with 650rpm shaking. The mixture was then centrifuged at 8000g for 8min at 4°C, and the top organic layer was collected and stored on ice. An additional 200ul of MTBE was added to the aqueous phase, vortex mixed for 30s, incubated at 4°C for 15min, and centrifuged at 8000g for 8min at 4°C. The organic layer was collected and combined with the previous extract. The collected organic layer was dried under N2 gas and re-suspended in 200ul of CHCl3/MeOH (1:1, v/v) containing 200uM of butylated hydroxytoluene (BHT) and stored at −20°C until analysis. Samples were further diluted with IPA/ACN/H2O (2:1:1 v/v) prior to analysis. The total protein content was measured with a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA) according to the manufacturer’s protocol, and used for the data normalization.

The liquid chromatography, tandem mass spectrometry (LC-MS/MS) analysis was conducted on an Ultimate 3000 Ultra High-Performance Liquid Chromatograph (UHPLC) coupled to a Thermo TSQ Altis Tandem Quadrupole Mass Spectrometer (Thermo Scientific, San Jose, CA). Chromatographic separation was achieved with an ACQUITY Amide BEH column (1.7μm, 2.1×100mm) maintained at 45°C (Waters, Milford, MO). Mobile phase compositions for solvents A and B consisted of ACN/H2O (95:5, v/v) and (50:50, v/v) respectively, with 10mM ammonium acetate. The gradient profile had a flow rate of 0.6ml min−1 and ramped from 0.1 to 20% B in 2min, from 20 to 80% B in 3min, dropped from 80 to 0.1% B in 0.1min, and held 0.1% B for 2.9min. Total chromatographic run time was 8.0min. The injection volume was 2ul. All analytes were detected using negative or positive ionization mode and selection reaction monitoring (SRM) library were adopted from Medina et al.72. The SRM is a targeted method that uses predetermined chromatographic retention times and precursor-to-production transitions to detect and identify each lipid, and has been implemented in our previously published studies73–75. Data were acquired using spiked-in internal standards (Avanti Polar Lipids EquiSPLASH, #330731) which provided one deuterium stable-labeled lipid per lipid class. The lipid standards allowed for compensation during lipid extraction and ionization efficiency. Collision energies and RF lens voltage were optimized for each reference standard. Data acquisition was performed using Xcalibur software v4.2 (Thermo Scientific, San Jose, CA) and processed using TraceFinder 5.1 (Thermo Scientific, San Jose, CA). Peak area was determined using ICIS peak peaking algorithm and 7-point smoothing. All peak area integration was manually verified. The peaks areas were then normalized to sum composition, mean-centered, and log-transformed using MetaboAnalyst 6.0 (https://www.metaboanalyst.ca/)76. The data as presented represent abundances rather than concentrations, enabling visualizing of hundreds of detected lipids with multivariate and univariate statistical analyses. Additional data analysis was done using MetaboAnalyst 6.0 and Prism 10 (GraphPad, La Jolla, CA). The lipidomics data generated in this study have been deposited in the Mendeley Data Repository under the https://doi.org/10.17632/nzwvkr7vgj.177.

Total Lipid Fatty Acid Analyses
Chemicals, reagents, and materials used for the lipid extractions and fatty acid analyses were of the highest quality available. MilliQ water, filtered deionized water is available in the laboratory. Methanol, optima grade was obtained from Thermo Fisher Scientific (Fair Lawn, NJ). Chloroform, HPLC grade was from Burdick & Jackson (Muskegan, MI), Stable-isotope-labeled internal standards (deuterated); were purchased from Cambridge Isotope Laboratories, Andover MA (internal standards C27:0 and C19:0; deuterium fatty acids, C8:0, 10:0, 12:0, 14:0, 15:0, 16, 18:0, 20:0, 22:0) or CDN Isotopes, Quebec, CAN (deuterium fatty acids C24:0, 26:0, pristanic and phytanic). Natural fatty acid standards were purchased from Nu Chek Prep (Elysian, MN) and were checked for purity by capillary gas chromatography by analysis on two capillary columns, a non-polar, DB1, and a polar, SP2560, capillary columns (50m×0.25mm×0.2µm) purchased from Supelco, Sigma-Aldrich, Inc, St. Louis, MO.

Frozen microdissected hippocampal tissues (total n=142; P11: male Nx n=4, IHx n=5, female Nx n=6, IHx n=5; P17: male Nx n=5, IHx n=6, female Nx n=6, IHx n=7; P22: male Nx n=6, IHx n=5, female n=6/group; P30: n=6/sex/group; P30 glycerol/glycerol-triacetate experiment-male: Nx glycerol n=6, Nx glycerol-triacetate n=5, IHx glycerol n=5, IHx glycerol-triacetate n=7; female: Nx glycerol n=6, Nx glycerol-triacetate n=6, IHx glycerol n=6, IHx glycerol-triacetate n=10; biological replicates) were weighed (0.1 to 0.2 g) and extracted with chloroform:methanol 2:1, v/v according to Folch et al.78. Total lipids were dried under N2 and weighed. The lipids were solubilized in 1ml chloroform:methanol 2:1, and approximately 1mg lipid from each sample was transferred to a 13x100mm screw capped test tube for analysis of total lipid fatty acids, including saturated, monounsaturated, and polyunsaturated of chain length C10 to C30. Fatty acids were hydrolyzed from triglycerides and phospholipids in a two-step process of acidification with 90:10v:v acetonitrile:6N HCl and neutralization with 90:10v:v methanol:10N NaOH, with incubations at 104°C for 45min, then reacidified in 6N HCl. Total fatty acids were extracted in hexane then derivatized with triethylamine and pentafluorobenzyl bromide (in acetonitrile) at room temperature 15min79,80. The resulting pentaflourobenzyl bromide esters were separated and quantified by capillary gas chromatography electron capture negative ion mass spectrometry (GCMS) by the method of Lagerstedt et al.81 except that a 50m×0.25mm×0.2µm SP2560 column was used for analysis. All fatty acids were analyzed on an Agilent 7890A GC coupled to a 5975C mass selective detector (MSD), with helium as the carrier gas and ammonia as the reagent gas. After a 1µl 1:35 split injection, the oven temperature was ramped more slowly at lower temperatures in two steps, resulting in a total run time of 55min. The initial oven temperature of 60°C was held for 2min, then increased at 10°C/min to 180°C, followed by a slower ramp at 2.5°C/min to 250°C, and then held for 14min. The MSD transfer line was initially maintained at 240°C for 33minutes, then increased at 2.5°C/min to a final temperature of 250°C and held for an additional 32min. Data were acquired in selection monitoring (SIM) mode, with dwell times ranging from 50ms to 100ms per ion species. Each analyte was matched to one of 13 stable-isotope labeled internal standards with the closest chain length and retention time. The fatty acids were quantitated using the Agilent MSD ChemStation software G1701EA version E.02.02.1431. The fatty acid retention time and peak integration were checked and each fatty acid was measured using a standard curve that was assayed with each set of analyses. For quality control, a reagent blank, a normal, and an abnormal plasma control samples were assayed with each set of analyses. Results were transferred into Excel for the final calculations as fatty acid ug/mg lipid weight.

Acylcarnitines
Acylcarnitines levels in the blood (total n=62; male: P11, P17, and P22 n=4/group; P30 Nx n=4, IHx, n=5; female: P11 Nx n=4, IHx n=3; P17 n=4/group; P22 Nx n=4, IHx, n=3; P30 Nx n=3, IHx, n=4; biological replicates) were quantified from dried blood spots (DBS) following a previously described method with modifications3,4. In brief, punched 1/8″ DBS samples were transferred into tubes containing 100ul of methanol and internal standards for acylcarnitines (NSKB; Cambridge Isotopes). Samples were incubated at 4°C for 20min and then dried under nitrogen. The dried samples were resuspended in 60μl 3N HCl in n-butanol and incubated at 65°C for 15min. Afterward, samples were dried under nitrogen and the residue containing butylated acylcarnitines was reconstituted in 100ul of mobile phase acetonitrile/water/formic acid (H2O:CH3CN:HCOOH; 80:19.9:0.1v/v%). Samples were vortexed, transferred to a centrifuge filter, and spun. The filtrates were then transferred to injection vials.

Acylcarnitines from frozen hippocampus (total n=63; male: P11, P17, and P22 n=4/group; P30 Nx n=4, IHx, n=5; female: P11 Nx n=5, IHx n=3; P17 n=4/group; P22 Nx n=4, IHx, n=3; P30 Nx n=3, IHx, n=4; biological replicates) were extracted by homogenizing the tissue in a solution of methanol containing the acylcarnitine standards. Samples were sonicated at room temperature for 10min and then centrifuged at 16,200×g for 4min at 4°C. After centrifugation, the liquid phase was transferred to a glass tube, evaporated under nitrogen, and processed as described above. Analysis of acylcarnitines was performed using an API 4500 mass spectrometer (AB SCIEX, Foster City, CA) operated in the positive-ion mode employing precursor ion scan for m/z 85, a characteristic production of butyl ester of acylcarnitine species. Data was processed using the ChemoView 2.0.2 (AB SCIEX) application. Blood samples are reported as nmol/ml and tissue samples as pmol/mg.

MALDI-MSI
MALDI MSI experiments were performed by the Johns Hopkins Applied Imaging Mass Spectrometry (AIMS) Core. All solvents and MALDI matrices were purchased from Sigma Aldrich (St. Louis, MO) and were used without further purification. All solvents were of HPLC grade or higher. Sample preparation: Brain samples from Nx and IHx P17 male mice were collected immediately after sacrifice and frozen using liquid nitrogen vapors (total n=8; n=4/group). Samples were stored at −80°C before cryosectioning. 10µm thick sagittal brain sections were collected using a Leica CM1860 UV Cryostat (Leica Biosystems, Wetzlar, Germany) and depth was verified by comparing tissue morphology to Allen Brain Atlas images at the target depth. Sections were thaw-mounted onto indium-tin oxide (ITO) coated glass microscope slides (Delta Technologys, Loveland, CO) and stored at −80°C prior to matrix application. Matrix application: Slides were warmed to room temperature in a vacuum desiccator, then sprayed with 5mg/mL α-cyano-4-hydroxycinnamic acid (CHCA) in 70% acetonitrile with 0.1% trifluoroacetic acid using an HTX M3+ pneumatic matrix sprayer (HTX Technologies, Chapel Hill, NC). Matrix was sprayed at 100µL/min for 8 passes using a 60°C nozzle temperature, 10psi nitrogen pressure, 1350mm/min nozzle velocity, 3mm track spacing, and 10second drying time. MALDI-MSI data acquisition: Immediately after matrix deposition, slides were imaged using a Bruker timsTOF fleX MALDI-2 mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with a SmartBeam 3D 10kHz Nd:YAG laser at 355nm with beam scan enabled and a 46um scan range. Height adjustment and laser focus tuning were performed prior to mass calibration. Mass calibration was performed using electrospray ionization (ESI) of Agilent ESI-L Tune Mix, resulting in a mass error <1 PPM. Instrument parameters were optimized to maximize intensity, as described in Supplementary Table 19. Data were acquired for m/z 100–950 in positive ion mode using a 50µm raster width with 1 burst of 225 laser shots per pixel. On-tissue MS/MS analysis was conducted by switching the instrument to MS/MS mode and collecting 5 summed bursts of 250 shots from the hippocampus of Nx and IHx samples for the parent and fragment spectra of each species of interest (Supplementary Figs. 10, 11). Parent ions were searched against the Lipid Maps database82–84. MALDI MSI data analysis: MALDI MSI data were imported into SCiLS Lab (version 2023b, Bruker Daltonics, Bremen, Germany). Data were normalized using total ion current (TIC) and analyzed using SCiLS lab’s feature finding and hypothesis testing workflows.

In-vivo metabolite uptake
Glucose uptake into the brain was measured using [1,2-3H]-2-Deoxy-D-glucose (2-DG) (Moravek Biochemicals; MT911), an unmetabolizable analog of glucose that enters cells but becomes trapped after phosphorylation. Mice were injected intraperitoneal (i.p.) with 0.2uCi/g of 2-DG diluted in sterile saline. Brains were harvested 45minutes after injection. Tissue from microdissected hippocampus was extracted using ice-cold 7% perchloric acid and centrifuged at 14,000g for 15min at 4°C. The supernatant was transferred to a vial containing scintillation fluid (National Diagnostics; LS-270) and radioactivity was measured in a LS 6500 Multi-purpose Scintillation Counter (Beckman Counter, Inc. USA). Disintegrations per minute (DPM) counts were normalized to wet tissue weight.

Fatty acid uptake into the hippocampus was measured using [1-14C]-2-Bromopalmitate (2-BP, Moravek Biochemicals; MC451). Mice were injected intraperitoneal (i.p.) with 0.2uCi/g of 2-BP complexed to bovine serum albumin and diluted in sterile saline. Microdissected hippocampus were harvested 45minutes after injection. Total lipids were extracted using the Folch method and transferred to vials containing scintillation liquid to measure radioactivity in a LS 6500 Multi-purpose Scintillation Counter (Beckman Counter, Inc. USA)78. DPM counts for 14C were normalized to wet tissue weight.

Ex-vivo oxidation studies
Oxidation assays were measured in stoppered tubes containing a center well with Whatman filter paper as previously described in refs. 3, 4, 85. In brief, the hippocampal region was microdissected and placed into tubes with labeling media containing either [1-14C]-oleic acid (Moravek Biochemicals; MC406), [U-14C]-D-glucose (Moravek Biochemicals; MC144W), [U-14C]-glutamine (Moravek Biochemicals; MC1124), [U-14C]-L-lactic acid (PerkinElmer; NEC599050UC), [3-14C]-D-3-Hydroxybutyric acid, (American Radiolabeled Chemicals, Inc; ARC#4122), or [1,2-14C]-acetic acid (PerkinElmer; NEC553050UC) and incubated in a 37°C water bath with gentle shaking for 3h. Labeling media was composed of 20% neurobasal medium (Gibco; 21103-049), 80% DMEM (Gibco; A14430), 0.5mM L-carnitine (Sigma; C0283), 0.2% bovine serum albumin (BSA; Sigma; A9647), and 25μM glutamine (Gibco; 25030). 100μM Etoximir (Sigma; E1905) was added to the media of the treated samples at the beginning of the assay as control for the [1-14C]-oleic acid oxidation assay. 14CO2 from complete substrate oxidation was trapped by the addition of 200μl of 1M perchloric acid in the medium and 150μl of 1M NaOH to the center well containing the filter paper and incubating the samples at 55°C for 1hour. The filter paper was transferred to a vial containing scintillation fluid and the radioactivity was counted 24h later. 1ml of acidified media was collected to measure acid-soluble metabolites, products from incomplete [1-14C]-oleic acid oxidation. 60μl of BSA and 100ul of 1M perchloric acid were added to the media and incubated overnight at 4°C. The next day, samples were centrifuged at 1467×g for 30min, the supernatant was transferred to vials containing scintillation fluid, and radioactivity was measured in a LS 6500 Multi-purpose Scintillation Counter (Beckman Counter, Inc. USA). DPM counts for 14CO2 and 14C-ASM were normalized to specific activity and wet tissue weight.

Lipid synthesis
Acetate incorporation into total lipid fraction was performed using [3H]-Acetate85,86. The microdissected hippocampal region was incubated at 37°C for 3h with 1uCi [3H]-Acetate (Moravek Biochemicals; MT634) in the same media composition used for the oxidation assays. Total lipids were extracted using the Folch method and transferred to vials containing scintillation fluid to measure radioactivity in a LS 6500 Multi-purpose Scintillation Counter (Beckman Counter, Inc. USA)78. DPM counts for 3H were normalized to wet tissue weight.

Data and statistical analysis
Data are shown as the mean ± SEM for each group unless otherwise stated. Data were analyzed using Prism 10 software (GraphPad, La Jolla, CA). Additional data analysis was performed using MetaboAnalyst 6.076. The statistical significance was determined using the 2-tailed Mann–Whitney test or unpaired Student’s t-test at instances where the total sample size was ≤7, 2-tailed paired t-test, and 2-way ANOVA with uncorrected Fisher’s LSD post-hoc. Statistical significance was set at p<0.05. Correlation analysis was performed using simple linear regression.

Artwork
Experimental design figures and biochemical pathways figures were created with BioRender.com full license plan.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "General comments:\nIn this study, Fernandez et al. investigated the impact of brain intermittent hypoxemia during neonatal stage on lipid\nmetabolism. Using a mouse model of neonatal brain injury due to intermittent hypoxemia, the authors reported that it led to\ncognitive impairments later in life. They also demonstrated that post-natal intermittent hypoxemia led to an alteration of lipid\nprofile signatured mainly by decreased levels of very long-chain fatty acids, which was associated with\nenhanced mitochondrial fatty acid oxidation. The authors also demonstrated that acetate treatment can promote functional recovery by\nsparing fatty acid oxidation.\nThe topic of investigation is novel and of high significance, the authors also performed a comprehensive study with logical\ndesign, which led to conclusions that could potentially be of clinical relevance. There are several considerations necessary\nto solidify their central conclusions, including verification of findings in human samples, justification of model used,\ndiscussion of potential sex differences, and interpretation of mechanism related to the altered lipid metabolism.\nMajor comments:\n1. The study has been performed in a mouse IHx model. Although the study timeline was well-designed, whether this model\nclosely resembles EPT neonatal hypoxia-induced brain injury demands further justification.\na) There are studies (e.g., PMID 33984336) reported that the age point as well as the interval of hypoxia treatment regimen\ncan lead to either impaired or improved memory function, the authors should provide literature support regarding the basis of\nIHx model set up in this study.\nb) Given the importance of time frame of IHx poses on the outcome, and the fact that IHx in human infant started preterm\n(EPT birth), this model is using a full term born mouse model to mimic post birth IHx, whether this could cause a difference in\nthe lipid or protein profile needs to be considered/discussed.\n2. The authors have demonstrated astrocyte as the major source of the increased fatty acid oxidation (FAO) after IHx using\nan astrocyte-specific CPT2 ko mouse model. This may align with the fact that astrocytes are the main cell for lipid\nmetabolism and function as a supporter for neurons. However, myelination is also in high demand during post-natal stage,\nand myelin is well-known to be highly-enriched in VLCFA. In Fig.1G, multiple top-altered lipid species are cerebrosides\n(Hex1Cer, Hex2Cer) that are specifically abundant in myelin sheaths. The possibility of potential contribution of\noligodendrocytes to the altered lipid profile under IHx condition should be re-evaluated and discussed.\n3. In concordance with Fig.1G which showed top lipid changes on P30, it would be informative to provide lipid species\nalteration data on P17 in Figure 3.\n4. As P11 is immediately after the IHx treatment, this would be a critical time point for any lipid change evaluation. In line\nwith this, Fig. S5B-D showed that P11 has a transient AC elevation in the IHx hippocampus. More comprehensive lipidomic\nanalysis should be provided at this time point, as it is important for understanding of events happened during and\nimmediately after IHx.\n5. P30 is still a relatively young age to make conclusion about potential long-term impact of IHx, lipid profile data from more\naged groups (with neonatal IHx) would be helpful to evaluate the significance of IHx impact.\n6. Data from Fig.1G showed that total CL (cardiolipin) levels were decreased in the IHx group. As CL plays a critical role in\nmitochondrial morphology, bioenergetics, dynamics, and signaling, have the authors noticed any differences between IHx\nand Nx in aspects such as mitochondrial number, quality, capacity, as well as tissue ATP levels (especially at P11)? These\nmeasurements and discussions will be important for interpreting findings of this study.\n7. The authors should be cautious in interpretating results from Fig. 3D. Data with sodium adduct in addition to potassium\nadduct should be provided to rule out the possibility of ion- vs. lipid- distribution resulted observation. Alternatively, this\nfigure could be removed.\n8. The authors have well-organized multiple aspects of their findings in main figures combining both sexes and displayed\nfurther sex-specific data in supplemental figures. While most of the findings showed similar trend between male and\nfemales, some observations show sex-specific patterns (e.g., Fig. S3B-C, Fig. S4C). Whether there are sex differences on\nthe long-term impact of EPT among humans, and whether the conclusions of this study have any sex discrepancy\nshould be discussed.\n9. Based on data from proteomics and mRNA levels (Fig.2C-D, and Fig. S7), it appears lipid metabolism (both fatty acid\nsynthesis and oxidation) pathways were down-regulated at P11 post IHx. Meanwhile, Mito Fuel functional test indicated\nincreased fatty acid oxidation (Fig. 3A). This contradiction should be further explained. The authors should be cautious in\ninterpretation of the [1-14C]-oleic acid as it only represents the oxidation of the first carbon (14C) and does not fully represent\nthe FAO activity overall.\n10. Fig. 2B showed both TCA and ETC proteins were down-regulated at P11, this is an understandable outcome of the\ndecreased oxygen availability during IHx. Considering that the shutdown of TCA and ETC itself may be sufficient to cause\naccumulation of intermediate metabolites of up-stream FAO, whether the enhanced AC levels at P11 is due to increased\nFAO activity, or due to shutdown of ETC is questionable, which leads an incomplete FAO and AC\naccumulation.\n11. The study in mouse model is comprehensive, yet the study lacks verification of its findings in human samples/models.\nConsidering the difficulty of acquiring human infant brain samples, some of the findings from this study could potentially be\nverified using samples from brain tissues of post-mortem infant who has experienced hypoxia (for example, hypoxic\nischemic encephalopathy infant cases are available from NIH NeuroBioBank). Also, some of the findings in blood samples\ncan be easily verified in human.\nMinor comments:\n1. Line 104 “reduction in fatty acids” does not seem to be accurate, as both reduction of very long-chain fatty acids (C22:0, C23:0,……),\nand increase of other fatty acids (C16:0, C20:4) were observed.\n2. The title should specify intermittent hypoxemia-induced neonatal brain injury, as neonatal brain injury may involve other\naspects/causes.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "Summary\nFernandez et al. extensively addressed an existing gap in the literature surrounding changes in brain lipid metabolism and\nthe brain lipid profile in infants born extremely preterm (i.e. less than 28 weeks gestation following brain injury). Authors used\nan established model of neonatal brain injury by subjecting mice to intermittent hypoxia (IHx) via episodes of hypoxia (32\ncycles) from P0-P11, alongside an arm of normoxia (Nx) controls. Male, and female pups from different litters\nwere used for the majority of experimentation at P11, P17, P22 and P30.\nAuthors examined the mouse hippocampus following injury and found that there was no change between IHx and Nx groups\nin total hippocampal lipids, however, changes in lipid composition were found that were in-part, sex-specific. Namely, at\nP30, males had significantly lower hippocampal omega 9 fatty acids (n-9), while palmitic acid (16:0; PAM) and arachidonic\nacid (20:4n-6; ARA) were significantly increased in IHx compared to Nx. In both sexes, very long chain fatty acids (VLCFA; >\nor equal to 20 carbons) were significantly decreased in IHx compared to Nx. Interestingly, PAM, the primary product of DNL\nwas the top differentially expressed fatty acid (FA) between IHx and Nx groups in males at P30.\nBased on changes in hippocampal lipid composition, authors sought to rule out if these changes were a result of altered\nnutrient supply following hypoxic injury. After measuring hippocampal glucose uptake, circulating NEFA levels, plasma\nketones and acyl-carnitines, and hippocampal acyl-carnitines, it was concluded that the change in hippocampal lipid\ncomposition was not a result of a limited supply of nutrients following injury. This led authors to conduct an unbiased\nproteomics analysis of the hippocampus at P11, in order to identify proteins and pathways responsible for the altered lipid\ncomposition following IHx. Postnatal day 11 was selected for proteomics because at this timepoint, plasma levels of ketones\nand acyl-carnitines were increased in IHx compared to Nx groups, and this corresponded to increased hippocampal acyl-\ncarnitines – giving insight to FAs being mobilized for the generation of acetyl-coA.\nAt P11, pathway analysis of proteomics data revealed the downregulation of multiple pathways involved in the metabolism\nof lipids in the IHx group; specifically, the downregulation of genes involved in FA oxidation (cpt2), the synthesis of long\nchain FA and cholesterol (scd2), lipid regulators (acc1), and FASN. However, these changes were transient and recovered\nby P30 (for example, FASN was downregulated at P11, and became upregulated by P30).\nAuthors then sought to address functional changes resulting from alterations in hippocampal lipid composition by assessing\nthe dependency of the hippocampus on different metabolic fuels. Using a seahorse mito fuel flex test kit; validated by\nmeasuring the oxidation of substrates labelled with 14C, authors found that metabolic brain fuels utilized depend on time.\nSpecifically, the injured brain depends more on the oxidation of glucose at P11 only, while FA oxidation was high at P11,\nand 6 days post injury (P17).\nBecause the hippocampal oxidation of FA remained high post-injury at P17, authors sought to determine if the brain also\nhad an increase in circulating FA uptake. Using an analogue of PAM that cannot be metabolized (3H-2-bromopalmitate),\nauthors found a significant increase in FA uptake in IHx mice compared to Nx, alongside unchanged 14C-glutamine, -BHB\nand -lactate uptakes at P17 between IHx and Nx. Authors concluded based on this result, that following\nhypoxia, from P11 to recovery (P17), the brain upregulates FA oxidation to fuel and support\noxidative metabolism.\nSince FA were extensively oxidized up until P17, authors then sought to measure the FA profile of the hippocampus at P17.\nIn both sexes, n-9 and total saturated fatty acids (SFAs) (> 20 carbons – which accumulate during development and are\nextensively synthesized) were significantly lower in IHx compared to Nx groups. Authors further characterized these\nchanges in situ using non targeted MS imaging in multiple brain regions, as well as in a conditional KO mouse of CPT2.\nTogether, these experiments showed IHx results in hippocampal region-specific lipid changes\n(corroborating the total lipid fatty acid profile at P17), and astrocytes are the primary brain cells\nthat oxidize FAs.\nLastly, and arguably most importantly, authors tested acetate treatment in a) the media and b) in vivo to investigate if acetate\nsupply would spare FA from oxidation (using oleic acid; 18:1n-9; OLA). Authors found treating the media with glucose to\nspare FA from oxidation did not impact OLA oxidation, however, OLA oxidation in the presence of acetate decreased by\n86% and 93% at P11, and P17, respectively, in IHx compared to Nx. Next, authors found in-vivo acetate treatment (glycerol-\ntriacetate to pups by oral gavage; 5mg/g birth weight) daily from P11-P17 reduced 14C-OLA oxidation by 71% at P17\ncompared to glycerol (vehicle) in the IHx group. Authors found in-vivo treatment with acetate also resulted in improved\nrecognition indexes in the Novel Object Recognition (NOR) test performed at the later timepoint of P30; suggesting an\nimprovement in recognition memory in addition to sparing FA from oxidation.\nAltogether, Fernandez et al. employed exhaustive approaches to investigate and characterize\nalterations following injury in the developing brain. As beta-oxidation is usually negligible in the developing brain, this data provides novel evidence that\nis not the case following neonatal injury, where beta-oxidation is significantly upregulated. This metabolic shift results in a\ndecreased VLCFA content in the brain, and interestingly, impacts cognitive performance (at P30) – in which\nsparing FA from oxidation can improve neurologic outcomes (the opposite to what is observed in\nadulthood, in which increased oxidation is associated with improved cognitive performance).\nImportantly, this paper demonstrates the unique specificity of the injured brain with regards to\nsubstrates utilized for oxidative metabolism during development, and results are promising for\nbeginning to investigate effective treatments following neonatal brain injury.\nSpecific comments:\nMajor comments:\n1. Palmitic acid is the primary product of de novo lipogenesis (DNL) and happens to be identified in males at P30 as the top\ndifferentially expressed FA in your volcano plot (Fig 1H; p < 0.001) between IHx and Nx groups, which is consistent with IHx\nmales having a significantly higher hippocampal PAM at P30 than Nx males. Could DNL be increasing in your IHx group in\nattempt to maintain brain energy requirements and/or FA balance in PL in response to injury? Especially since FASN (the\nmajor governor of DNL) was also upregulated at P30 in IHx males? Not a lot of discussion/hypotheses around this result,\nwhich also appears to be sex-specific.\n2. Almost all results shown are limited to the hippocampus, and not the whole brain (other than the MS imaging data). How\nmight you predict your findings to change at the level of the whole brain, or other regions? If hippocampus was important a\npriori this could be mentioned.\n3. Authors state there is a “reduction” in brain lipids in the abstract and elsewhere, however, total lipids in the hippocampus\n(primary region examined) are unchanged between IHx and Nx groups (% of total lipids per mg of tissue). Rather, it is lipid\ncomposition which changed. Language needs to be 1) altered to reflect this is not a real “reduction”, and 2) clarify this result\nis region-specific.\n4. Presenting hippocampal lipid composition data in a supplemental table – as both concentration and relative %,\nwould be useful to the reader.\n5. There is an overall lack of justification surrounding why the hippocampus was selected for analyses (see comment\nabove), besides the obvious reason it corresponds to behavioural analyses conducted. Is the hippocampus equipped for\noxidation compared to others? Lipid synthesis? This could be explained better.\n6. Pups were removed from the dam for extended periods of time very early in development to undergo cycles of hypoxia (16\nx 3 min hypoxia + 5 min re-oxygenation x 2 = 32 cycles). This is a significant amount of time, and enough to impact maternal\nstress. Did authors measure and/or consider maternal stress, and if this can program pups postnatally?\n7. This is a lot of assays for a very small brain tissue to undergo. A supplemental table outlining which pups underwent\nwhich assay would aid in the reader understanding the results better. Furthermore, the use of “mice” throughout the methods\nshould specify the age, and sex of the mice as authors used many timepoints and both sexes.\n8. Were any other tissues collected from these mice? It would be nice to see the FA composition of a peripheral tissue like\nthe liver and rule out if total lipids changed in peripheral tissues, but not the brain. Perhaps there is between-tissue FA\nexchange to maintain total brain FAs.\n9. IHx group weighs significantly less than Nx at most timepoints in both sexes. I understand this is necessary to model\nextremely preterm infants, however, this could also impact overall metabolism, and substrates used for energy. Are\nhippocampal lipid alterations a result of hypoxia, weight loss, or both?\nMinor comments:\n1. Abstract: The abstract overall is a bit boilerplate for the results described. Adding more detail to the results with regards to\ntimepoints, sex, etc. would better capture your findings.\n2. Abstract: Line 109: “timely” supply – again, we know this is a critical period of brain development but what exactly are the\ntimepoints that the intervention (triacetin) would have the most benefit?\n3. Introduction: line 143: provide a citation for IHx as clinically relevant model of neonatal brain injury?\n4. Introduction: line 147: “clinically feasible source of acetate” – provide more detail supporting why this is clinically feasible?\nI assume it has a bitter taste; it is recognized safe as a food additive but what dose would it need to be administered as, and\nwould this be GRAS?\n5. Language around timepoints selected and interpretation:\na. Line 153-154: the “neonatal period” authors selected not only impacts what would be a premature infant (PND 1-2/3 in a\nmouse), but also the window of a term infant (PND 7-10 mouse) – translationally, are children that are born extremely\npreterm exposed to intermittent hypoxia as a term infant, as well?\nb. By P30 in a mouse, the brain is 90-95% of adult weight and corresponds to what would be a 2-3 year old child. Be careful\nwith language around this timepoint referring to “later” in life.\n6. Figure 1B-D and Extended Figure 1A-B: Add units for x-axis (i.e. “day 30” or “P30”). It is unclear what units you are\nreferring to.\n7. Figure 1E-F: Inconsistency in naming of PC 1/2 – pick either PC or component.\n8. I would consider adding the differentially expressed fatty acid data for females into the figure with males, as it is equally as\nimportant and tells the whole story.\n9. Extended data Figure 1C – are IHx males resistant to changes in body weight at 17 days? Or is this driven by variability,\netc.?\n10. Extended Figure 1C,D and elsewhere: Please clarify “age” on x-axis as number of days.\n11. Results: line 169-170: the primary substrate in humans and in rodents for DNL is glucose, not other fatty acids (they do\nindeed contribute but not to a great extent). Work has demonstrated this, in the brain, during development:\nhttps://www.nature.com/articles/s41467-023-44388-4 (I see you cite this already later on as well – it should be mentioned\nhere for consistency).\n12. Results: Line 180-182: “prolonged dysregulations in lipid homeostasis” – what kind of dysregulations?\nIncreased/decreased DNL? Increased/decreased oxidation? A bit of an oversimplified statement for your detailed results.\n13. The concept of astrocytes being specialized to oxidize fatty acids is well-established (for example:\nhttps://onlinelibrary.wiley.com/doi/10.1111/gtc.13144) – is your finding novel in the timepoints/model selected? What makes\nyour finding novel?\n14. Discussion is somewhat brief – more comparisons to the existing literature could be made on this topic.\n15. What kind of diets were the pups on post-weaning, or the dams throughout the study? This is not mentioned in the\nmanuscript.\n16. Were the mice bred in-house, or were the dams ordered pregnant? This is not clear.\n17. What methods were used for randomization? (line 405, line 419)\n18. Who conducted the behavioural tests, as well as most analyses? Were investigators blinded or unblinded to treatment?\n19. Clarify what portion of the brain was used for lipidomics (line 516) – was this from the same brains that the hippocampus\nwas taken from? Left vs. right hemisphere? Remaining whole brain?\n20. Sample size is listed as a range in figure legends (ex. 7-13/sex/group/treatment) – as per Nature Comm requirements,\nsample size must specify the total number of mice per sex, per treatment and not be presented in a range format.\n21. Sample size for NOR test is rather small, is this enough power to detect changes?\n22. Were the RT PCR data validated?\n23. Authors refer to VLCFA as FA > 20 carbons – is it 20 or 22 that deems them VLCFA? Check IUPAC\n24. Lines 187-189, you state the developing brain uses fatty acids for brain lipid synthesis, then follow it with the developing\nbrain extensively using glucose for de novo synthesis of fatty acids – these statements contradict each other.\n25. As per Nature Comm requirements “as previously described” must be replaced with exactly how the procedure was\ncarried out (line 661) in the methods sections\n26. Blood measurements in pups – these were unfasted or fasted?", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_5": "The manuscript by Fernandez et al. provides new evidence that during the critical period of brain development in neonates\nborn extremely prematurely, brain mitochondria increasingly rely on fatty acid oxidation. This metabolic shift coincides with a\nreduction in total brain fatty acids and complex lipid classes, which may contribute to behavioral changes. The authors\ndemonstrate that supplementation with alternative fuels, such as acetate, in in vivo and in vitro models, spares fatty\nacids and leads to improved neurological outcomes. Authors use of intermittent hypoxia as a model to study neonatal brain injury\nin extremely preterm infants. Overall, the manuscript is well presented, and the major conclusions are supported by the data.\nThe reviewer suggests the following improvements to enhance the clarity and rigor of the manuscript:\n1. Authors need to improve the consistency in Definitions of Fatty Acid Classes. In line 355, the authors consider very long-\nchain fatty acids (VLCFA) as lipids with more than 22 carbons; however, in Figures 1J/K (and similarly in Extended Figures\n2C/D, 9B/C), the cutoff appears to be 20 carbons. Can the authors reconcile this discrepancy between the results and\nconclusions? For consistency, it is generally accepted that long-chain fatty acids (LCFA) include lipids with 12–20 carbons,\nwhile VLCFA are those with 22–38 carbons. Distinguishing between these two categories is important, as the data treat all\nfatty acids similarly, yet the discussion focuses primarily on VLCFA.\n2. Presentation of Lipid Remodeling Data could be also improved. The changes shown in lipid remodeling of free fatty acids\nappear modest (Figures 1I, Extended Figures 2B, 4F, 5D, 3C, and 9A), and the graphs make it difficult to discern statistically\nsignificant trends. Rather than displaying all data, including non-significant changes, the authors could provide a\ncomprehensive table summarizing all results and only depict statistically significant changes in the figures for clarity.\n3. Could the observed phenotype be explained simply by low body weight and, consequently, a reduced capacity to\naccumulate adipose tissue, which is essential for normal brain development? The authors are encouraged to address this\npossibility.\n4. The authors report that supplementation with glycerol-triacetate improves neurological recovery based on NOR scores.\nDoes this treatment also result in improvements in brain lipidomic profiles, particularly in the hippocampus? Including this\ndata and more presenting more phenotypic data would strengthen the conclusions.\nMinor Suggestions:\n- The term “differentially expressed lipids” is somewhat misleading. Lipids, unlike genes, may not “express” information or\nfunctions in the traditional sense; many serve structural roles, such as membrane components. The authors are encouraged\nto clarify or replace this terminology.\n- Extended Figure 1C-D: It would be valuable to include pre-intervention data as a control to confirm that both groups started\nwith comparable body weights.\n- Extended Figure 4: The data likely represent fed-state levels; the authors should clarify this in the figure caption."}, "author_response": "REBUTTAL LETTER\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nGeneral comments:\nIn this study, Fernandez et al. investigated the impact of brain intermittent hypoxemia during\nneonatal stage on lipid metabolism. Using a mouse model of neonatal brain injury due to\nintermittent hypoxemia, the authors reported that it led to cognitive impairments later in life. They\nalso demonstrated that post-natal intermittent hypoxemia led to an alteration of lipid\nprofile signatured mainly by decreased levels of very long-chain fatty acids, which was associated with\nenhanced mitochondrial fatty acid oxidation. The authors also demonstrated that acetate\ntreatment can promote functional recovery by sparing fatty acid oxidation.\nThe topic of investigation is novel and of high significance, the authors also performed a\ncomprehensive study with logical design, which led to conclusions that could potentially be of\nclinical relevance. There are several considerations necessary to solidify their central conclusions,\nincluding verification of findings in human samples, justification of model used, discussion of\npotential sex differences, and interpretation of mechanism related to the altered lipid metabolism.\nMajor comments:\n1. The study has been performed in a mouse IHx model. Although the study timeline was well-\ndesigned, whether this model closely resembles EPT neonatal hypoxia-induced brain injury\ndemands further justification.\na) There are studies (e.g., PMID 33984336) reported that the age point as well as the interval of\nhypoxia treatment regimen can lead to either impaired or improved memory function, the authors\nshould provide literature support regarding the basis of IHx model set up in this study.\nBased on numerous clinical and preclinical studies, intermittent hypoxia due to apnea of\nprematurity is detrimental to neurodevelopmental outcomes (PMID: 26262797, PMID: 15329741,\nPMID: 25476492, and PMID: 28388601)1–4. Hyperoxia also has been found to be detrimental and\ntherefore, in the current clinical practice, titration of oxygen in extremely preterm infants is\ncommon. In these patients, hyperoxia has been shown to contribute to other morbidities such as\nretinopathy of prematurity (ROP), therefore currently, clinical practice aims to limit hyperoxia\n(PMID: 31487704 and PMID: 20689332)5,6.\nIn the review paper that the reviewer cites (PMID: 33984336)7, intermittent hypoxia was used to\ncounterbalance hyperoxia and hyperoxia detrimental pathways. This review cites the works of\nBavis et al. (2019) (PMID 30439529)8 which explored carotid body development and brain stem\nhypoxia ventilator response in a hyperoxia model during the first 12 days of postnatal\ndevelopment. This paper (PMID 30439529)8 found that IHx improves carotid body development\nbut did not explore brain development. The other cited paper in this review article from Martin et\nal. (2010)9 used a model of neonatal asphyxia with a single 5-min exposure to 100% N 24 hours\n2\nafter birth followed by oxygen resuscitation. These two papers model different clinical paradigms\nwhich are not related to apnea of prematurity. Our model of intermittent hypoxia recapitulates\napnea of prematurity which is characterized by multiple daily brief episodes of hypoxia with a rapid\nreturn to normoxia (PMID: 33597229 and PMID: 33712716)10,11. This in clinical patients can\ncontinue until age equivalent term.\nThe papers from Zhang et al. (2005)12 titled “Neonatal exposure to intermittent hypoxia enhances\nmice performance in water maze and 8-arm radial maze tasks” and Zhang et al. (2014) (PMID:\n24368168)13 sought to understand high altitude (2km) associated hypoxia effects on brain\ndevelopment. Their intermittent hypoxia model consists of one 4-hr long exposure to 16% O daily\n2\nand does not represent brief repetitive episodes common to apnea of prematurity. Our study, in\ncontrast, aimed to understand the effects of multiple and brief episodes of hypoxia which last 20\nseconds or longer and associated with low oxygen saturations. As clinicians (Susanna Scafidi,\nMD – a pediatric ICU physician and Joseph Scafidi, DO – a neonatal neurologist and\nneurodevelopmental specialist), we care for children born extremely preterm that experienced\napnea of prematurity and subsequent co-morbidities. Therefore, we utilized a model that mimics\nour clinical experience and experience of other clinician scientists that are trying to address the\nsequela of intermittent hypoxemia due to extremely preterm birth. Specifically, Vadim Ten, MD\n(neonatologist) research group has shown that this model reflects neurologic, histologic, and\npathophysiologic changes observed in humans born extremely preterm (PMID: 25476492)3.\nOther scientists such as Dr. Alexander Drobyshevsky’s research group also utilizes a similar\nmodel of intermittent hypoxia to study the cellular effect on brain development (PMID: 39890465\nand PMID: 30858028)14,15.\nb) Given the importance of time frame of IHx poses on the outcome, and the fact that IHx in human\ninfant started preterm (EPT birth), this model is using a full term born mouse model to mimic post\nbirth IHx, whether this could cause a difference in the lipid or protein profile needs to be\nconsidered/discussed.\nWe agree with the reviewer that timing is critical during brain development. It has been established\nthat the rodent’s first 8-10 days (Postnatal days 0-10) of postnatal life have biological features\nsimilar to human brains in the 3rd trimester of gestation. Numerous original research studies\ndelineate different aspects of brain development in humans and rodents. Below is a list of several\nreviews that summarize the pertinent findings:\nPMID: 2358330716\nPMID: 1531333417\nPMID: 3324657818\nPMID: 3456937519\nIn fact, the emergence of new technology has enabled researchers to obtain more granular and\nsophisticated data to correlate similar features between humans and other species such as\nrodents. Newer generated data further confirms the choice of age in our model (PMID:\n39870528)20.\nIn the revised version of the manuscript, we include citations that report “the sequence of key\nevents in brain maturation is largely consistent between humans and rodents” during development\n(PMID: 23583307)16.\n2. The authors have demonstrated astrocyte as the major source of the increased fatty acid\noxidation (FAO) after IHx using an astrocyte-specific CPT2 ko mouse model. This may align with\nthe fact that astrocytes are the main cell for lipid metabolism and function as a supporter for\nneurons. However, myelination is also in high demand during post-natal stage, and myelin is well-\nknown to be highly-enriched in VLCFA. In Fig.1G, multiple top-altered lipid species are\ncerebrosides (Hex1Cer, Hex2Cer) that are specifically abundant in myelin sheaths. The possibility\nof potential contribution of oligodendrocytes to the altered lipid profile under IHx condition should\nbe re-evaluated and discussed.\nWe thank the reviewer for the insightful question. Altered sphingolipids are associated with\ninflammation, autophagy, cell growth and survival (PMID: 26703189, PMID: 26943039, and\nPMID: 22230369)21–23. In this study, the lipidomic profile was performed on microdissected\nhippocampus, thus, several cell types can be affected (PMID: 32937123)24. Our lipidomic profile\nhas shown that at P30 (19 days post-IHx) several Hex1Cer and Hex2Cer species were both down-\nand up-regulated (See table below). Indeed, oligodendrocytes, which contain high levels of\nHexCer, can be affected in this model and we are currently exploring this further specifically in\nmicrodissected subcortical white matter. As noted in the review “Lipid metabolism in astrocytic\nstructure and function” by Lee et al. (2021)25, changes to astrocyte sphingolipid structure and\nmetabolism have been described in various neurodegenerative diseases (PMID: 15590150,\nPMID: 22810490, and PMID: 30803453)26–28. Specifically, astrocyte production of cerebrosides\n(HexCer) as precursors to gangliosides supports healthy neurite outgrowth, regulates neuronal\ninflammation, and stabilizes neuron-glia interactions (PMID: 31724708)29.\nRecently, McInnis et al. (2024) (PMID: 39616264)30 reported that human iPSCs derived neurons\nand glia respond differently to ceramides. Thus, the detection of altered sphingolipids during\ndevelopment between Nx and IHx supports that sphingolipids play a pivotal role in cellular\nphysiology including development and after intermittent hypoxemia.\nTable showing changes in Hex1Cer and Hex2Cer in IHx-injured hippocampus relative to Nx\ncontrols at P30:\nLipid Fold change Log2 (Fold Change) Raw (p-value) -Log10 (p-value)\nHex1Cer d18:1/24:1 0.00019 -12.384 0.001 3.079\nHex1Cer d18:1/26:1 0.00017 -12.538 0.001 2.9735\nHex1Cer d18:1/26:0 0.00024 -12.024 0.002 2.7503\nHex1Cer d18:1/22:1 0.0293 -5.0909 0.018 1.7383\nHex1Cer d18:1/18:1 5619.00 12.456 0.004 2.4491\nHex1Cer d18:2/22:0 4037.70 11.979 0.004 2.4349\nHex1Cer d18:2/20:0 6502.20 12.667 0.004 2.4056\nHex1Cer d18:1/14:0 14057.00 13.779 0.005 2.336\nHex1Cer d18:1/18:0 3957.70 11.95 0.033 1.483\nHex1Cer d18:1/16:0 12.28 3.618 0.031 1.5037\nHex2Cer d18:1/18:0 10781.00 13.396 0.002 2.7173\nHex2Cer d18:1/20:0 35.60 5.1539 0.004 2.4031\nHex2Cer d18:1/ 22:0 8576.00 13.066 0.012 1.9258\nHex2Cer d18:1/16:0 44.12 5.4632 0.016 1.8027\nHex2Cer d18:1/24:2 3.79 1.9217 0.025 1.61\n3. In concordance with Fig.1G which showed top lipid changes on P30, it would be informative to\nprovide lipid species alteration data on P17 in Figure 3.\nWe thank the reviewer for this suggestion. We have expanded this study to now include lipidomic\nprofile of the microdissected hippocampus at P11 (Figure 3), P17 (Figure 6) and P22\n(Supplementary Figure 8).\n4. As P11 is immediately after the IHx treatment, this would be a critical time point for any lipid\nchange evaluation. In line with this, Fig. S5B-D showed that P11 has a transient AC elevation in\nthe IHx hippocampus. More comprehensive lipidomic analysis should be provided at this time\npoint, as it is important for understanding of events happened during and immediately after IHx.\nWe thank the reviewer for this suggestion. As stated in comment #3 above, we have now included\nthe lipidomic profile at P11 (Figure 3).\n5. P30 is still a relatively young age to make conclusion about potential long-term impact of IHx,\nlipid profile data from more aged groups (with neonatal IHx) would be helpful to evaluate the\nsignificance of IHx impact.\nWe agree with the reviewer that the sequalae of IHx can be long-lasting. In this study, we aimed\nto address the current gaps in knowledge regarding brain maldevelopment in school age children.\nA majority of our patients (JS) born extremely preterm return and/or present for neurological and\nbehavioral re-assessment at school age due to inability to ‘keep up’ with academic performance\ncompared to their peers. As we stated in response #1 above, murine P30 age correlates to\nchildhood – school age. Therefore, we focused on this specific age group. Future studies will\naddress young adulthood and aging in this model of extremely preterm brain injury.\n6. Data from Fig.1G showed that total CL (cardiolipin) levels were decreased in the IHx group. As\nCL plays a critical role in mitochondrial morphology, bioenergetics, dynamics, and signaling, have\nthe authors noticed any differences between IHx and Nx in aspects such as mitochondrial\nnumber, quality, capacity, as well as tissue ATP levels (especially at P11)? These measurements\nand discussions will be important for interpreting findings of this study.\nWe thank the reviewer for this astute and thoughtful comment. Indeed, we were similarly surprised\nby the profound changes in CL species that were present 19 days post-IHx injury. With inclusion\nof new lipidomics data from P11, P17 and P22, we are able to show that these changes of CL\nincreased with age. Our lab is currently pursuing this further. In the interim, we assessed\nmitochondrial content by quantifying mitochondrial to nuclear DNA ratio using digital PCR and this\ndata is now included in Figure 4 and Supplementary Figure 5. At P30, where CL species were\nlargely decreased, the mitochondrial to nuclear DNA ratio was not different. The premise of these\nexperiments were based on Martin Picard group’s recent publications (PMID: 40140564 and\nPMID: 37563104)31,32.\n7. The authors should be cautious in interpretating results from Fig. 3D. Data with sodium adduct\nin addition to potassium adduct should be provided to rule out the possibility of ion- vs. lipid-\ndistribution resulted observation. Alternatively, this figure could be removed.\nWe thank the reviewer for this comment. We agree with the reviewer and we provide the spectra\nand image with the sodium adduct (Supplementary Figure 11). This revised manuscript contains\nboth sodium and potassium adduct. We included this additional data into the manuscript. This\ndata reassures us that the observed changes in the lipids using MALDI-MSI is not due to the\nsodium or potassium adducts.\n8. The authors have well-organized multiple aspects of their findings in main figures combining\nboth sexes and displayed further sex-specific data in supplemental figures. While most of the\nfindings showed similar trend between male and females, some observations show sex-specific\npatterns (e.g., Fig. S3B-C, Fig. S4C). Whether there are sex differences on the long-term impact\nof EPT among humans, and whether the conclusions of this study have any sex discrepancy\nshould be discussed.\nThank you for this comment. In the results and discussion, we point out sex differences at the\ndifferent age groups as well as discussion point about sex differences in humans.\n9. Based on data from proteomics and mRNA levels (Fig.2C-D, and Fig. S7), it appears lipid\nmetabolism (both fatty acid synthesis and oxidation) pathways were down-regulated at P11 post\nIHx. Meanwhile, Mito Fuel functional test indicated increased fatty acid oxidation (Fig. 3A). This\ncontradiction should be further explained. The authors should be cautious in interpretation of the\n[1-14C]-oleic acid as it only represents the oxidation of the first carbon (14C) and does not fully\nrepresent the FAO activity overall.\nWe thank the reviewer for pointing this fact out. Changes in gene and protein expression often\nrequire assessment of function by measuring their activity and/or product/intermediate produced\nby this particular protein. It is generally accepted that increased protein expression is associated\nwith increased function. However, existing proteins might carry out activity very efficiently even at\nthe small levels thus generating negative feedback loop which results in post-translational\nchanges, i.e. reduced transcription or increased degradation of mRNA. Similarly, decreased\nactivity may trigger increase in mRNA expression and subsequent protein increase. Several\nrecent studies, albeit in other tissues (liver, muscle), explored the discordance between the levels\nof mRNA, protein, and activity (PMID: 40376090)33. Caution and thorough experimental\nevaluation is underscored in a recent article by Lee et al. in Nature Metabolism (PMID:\n40211046)34. Our approach in this study was to comprehensively assess gene and protein\nexpression as well as function (Seahorse, oxidation studies and quantification of metabolites).\nAs pointed out by the reviewer, we used [1-14C]-oleic acid to assess β-oxidation of fatty acids\nsince there is no [U-14C]-oleic acid available. Using [1-14C]-oleic acid, we are measuring the first\ncycle, which contains the activation and transport steps involving the β-oxidations enzymes, which\nis the rate-limiting cycle. We are measuring oxidation of the same fatty acid under different\nconditions. In addition, we also included in the manuscript the acid soluble metabolites (ASMs)\n(see Supplementary Figures 7 and 9). Our data demonstrates changes in CO production without\n2\nchanges in ASMs. This signifies that β-oxidation of the fatty acid is near-complete.\n10. Fig. 2B showed both TCA and ETC proteins were down-regulated at P11, this is an\nunderstandable outcome of the decreased oxygen availability during IHx. Considering that the\nshutdown of TCA and ETC itself may be sufficient to cause accumulation of intermediate\nmetabolites of up-stream FAO, whether the enhanced AC levels at P11 is due to increased\nFAO activity, or due to shutdown of ETC is questionable, which leads an incomplete FAO and AC\naccumulation.\nThank you for this comment. We would like to clarify that under this paradigm of IHx (8% decrease\nO with re-oxygenation), there is oscillating levels of oxygen but not necessarily shutdown of TCA\n2\nand ETC. Rather, there is decreased expression of ETC proteins (proteomics data), and\nmitochondrial content (mtDNA/nDNA ratio at P11). At the same time point (P11), there is increase\noxidation of glucose, sustained oxidation of glutamine, and increased oxidation of fatty acids.\nIncreased acylcarnitines in the hippocampal dissected tissue reflects mobilization of fatty acids\ntowards mitochondrial β-oxidation and/or generation of ketones as reflected in our ASMs levels.\nIn our data, we extensively interrogated the oxidation of multiple substrates including glucose,\nglutamine, βHB, and lactate and demonstrated that oxidation still took place following IHx.\n11. The study in mouse model is comprehensive, yet the study lacks verification of its findings in\nhuman samples/models. Considering the difficulty of acquiring human infant brain samples, some of\nthe findings from this study could potentially be verified using samples from brain tissues of\npost-mortem infant who has experienced hypoxia (for example, hypoxic ischemic encephalopathy\ninfant cases are available from NIH NeuroBioBank). Also, some of the findings in blood samples\ncan be easily verified in human.\nWe do agree with this reviewer that our pursuit of a mouse study was to understand human\npathology. We appreciate that this reviewer understands the difficulties of obtaining human brain\ntissue at school age. While blood samples might be easier to obtain, logistically it will require IRB,\npatient recruitment, and specific biomarker analysis. As we demonstrate in this manuscript, many\nprocesses occur specifically in the brain, and blood levels did not reflect brain changes in\ncirculating fatty acids, acylcarnitines, and other metabolites.\nDiagnosis of hypoxic-ischemic encephalopathy represents a different diagnosis and pathological\nprocess. Specifically, newborns (both term and any gestational age pre-term) will sustain acute\nsingle episode of asphyxia/hypoxia and subsequent ischemia often due to umbilical cord wrap\naround the neck at the time of delivery or decrease in focal delivery. In term neonates, the extent\nof immediate injury is determined clinically by the Sarnat scoring, however, this scoring does not\napply to preterm neonates. In contrast, nearly 100% of extremely preterm newborns have\nnumerous episodes of apnea of prematurity, associated with brief low oxygen levels and these\nepisodes continue during the entire time till infants reach corrected full-term age.\nWe did contact the NIH NeuroBioBank as per reviewer suggestion. There were no human\nsamples with history of prematurity at school age. We were able to get a small cohort of human\nsamples for both term (control) and extremely preterm (<28 weeks of gestation) infants. We\nperformed total lipid fatty acid analysis, which is now presented in Supplemental Figure 2. These\nresults are comparable to mouse brain tissue total lipid fatty acid profile at P11.\nMinor comments:\n1. Line 104 “reduction in fatty acids” does not seem to be accurate, as both reduction of very long-\nchain fatty acids (C22:0, C23:0,……), and increase of other fatty acids (C16:0, C20:4) were\nobserved.\nThis was corrected for “Our results show changes in hippocampal lipid composition and\nabnormal fatty acid profile”.\n2. The title should specify intermittent hypoxemia-induced neonatal brain injury, as neonatal brain\ninjury may involve other aspects/causes.\nTitle amended for the following: “Dysregulated Fatty Acid Metabolism following Intermittent\nHypoxemia-induced Neonatal Brain Injury is Rescued by Treatment with Acetate”\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #3 (Remarks to the Author):\nSummary\nFernandez et al. extensively addressed an existing gap in the literature surrounding changes in\nbrain lipid metabolism and the brain lipid profile in infants born extremely preterm (i.e. less than\n28 weeks gestation following brain injury). Authors used an established model of neonatal brain\ninjury by subjecting mice to intermittent hypoxia (IHx) via episodes of hypoxia (32 cycles) from\nP0-P11, alongside an arm of normoxia (Nx) controls. Male, and female pups from different litters\nwere used for the majority of experimentation at P11, P17, P22 and P30.\nAuthors examined the mouse hippocampus following injury and found that there was no change\nbetween IHx and Nx groups in total hippocampal lipids, however, changes in lipid composition\nwere found that were in-part, sex-specific. Namely, at P30, males had significantly lower\nhippocampal omega 9 fatty acids (n-9), while palmitic acid (16:0; PAM) and arachidonic acid\n(20:4n-6; ARA) were significantly increased in IHx compared to Nx. In both sexes, very long chain\nfatty acids (VLCFA; > or equal to 20 carbons) were significantly decreased in IHx compared to\nNx. Interestingly, PAM, the primary product of DNL was the top differentially expressed fatty acid\n(FA) between IHx and Nx groups in males at P30.\nBased on changes in hippocampal lipid composition, authors sought to rule out if these changes\nwere a result of altered nutrient supply following hypoxic injury. After measuring hippocampal\nglucose uptake, circulating NEFA levels, plasma ketones and acyl-carnitines, and hippocampal\nacyl-carnitines, it was concluded that the change in hippocampal lipid composition was not a result\nof a limited supply of nutrients following injury. This led authors to conduct an unbiased\nproteomics analysis of the hippocampus at P11, in order to identify proteins and pathways responsible for the\naltered lipid composition following IHx. Postnatal day 11 was selected for proteomics because at\nthis timepoint, plasma levels of ketones and acyl-carnitines were increased in IHx compared to\nNx groups, and this corresponded to increased hippocampal acyl-carnitines – giving insight to\nFAs being mobilized for the generation of acetyl-coA.\nAt P11, pathway analysis of proteomics data revealed the downregulation of multiple pathways\ninvolved in the metabolism of lipids in the IHx group; specifically, the downregulation of genes\ninvolved in FA oxidation (cpt2), the synthesis of long chain FA and cholesterol (scd2), lipid\nregulators (acc1), and FASN. However, these changes were transient and recovered by P30 (for\nexample, FASN was downregulated at P11, and became upregulated by P30).\nAuthors then sought to address functional changes resulting from alterations in hippocampal lipid\ncomposition by assessing the dependency of the hippocampus on different metabolic fuels. Using\na seahorse mito fuel flex test kit; validated by measuring the oxidation of substrates labelled with\n14C, authors found that metabolic brain fuels utilized depend on time. Specifically, the injured\nbrain depends more on the oxidation of glucose at P11 only, while FA oxidation was high at P11,\nand 6 days post injury (P17).\nBecause the hippocampal oxidation of FA remained high post-injury at P17, authors sought to\ndetermine if the brain also had an increase in circulating FA uptake. Using an analogue of PAM\nthat cannot be metabolized (3H-2-bromopalmitate), authors found a significant increase in FA\nuptake in IHx mice compared to Nx, alongside unchanged 14C-glutamine, -BHB and -lactate\nuptakes at P17 between IHx and Nx. Authors concluded based on this result, that following\nhypoxia, from P11 to recovery (P17), the brain upregulates FA oxidation to fuel and support\noxidative metabolism.\nSince FA were extensively oxidized up until P17, authors then sought to measure the FA profile\nof the hippocampus at P17. In both sexes, n-9 and total saturated fatty acids (SFAs) (> 20 carbons\n– which accumulate during development and are extensively synthesized) were significantly lower\nin IHx compared to Nx groups. Authors further characterized these changes in situ using non\ntargeted MS imaging in multiple brain regions, as well as in a conditional KO mouse of CPT2.\nTogether, these experiments showed IHx results in hippocampal region-specific lipid changes\n(corroborating the total lipid fatty acid profile at P17), and astrocytes are the primary brain cells\nthat oxidize FAs.\nLastly, and arguably most importantly, authors tested acetate treatment in a) the media and b) in\nvivo to investigate if acetate supply would spare FA from oxidation (using oleic acid; 18:1n-9;\nOLA). Authors found treating the media with glucose to spare FA from oxidation did not impact\nOLA oxidation, however, OLA oxidation in the presence of acetate decreased by 86% and 93%\nat P11, and P17, respectively, in IHx compared to Nx. Next, authors found in-vivo acetate\ntreatment (glycerol-triacetate to pups by oral gavage; 5mg/g birth weight) daily from P11-P17\nreduced 14C-OLA oxidation by 71% at P17 compared to glycerol (vehicle) in the IHx group.\nAuthors found in-vivo treatment with acetate also resulted in improved recognition indexes in the\nNovel Object Recognition (NOR) test performed at the later timepoint of P30; suggesting an\nimprovement in recognition memory in addition to sparing FA from oxidation.\nAltogether, Fernandez et al. employed exhaustive approaches to investigate and characterize\nalterations following injury in the developing brain. As beta-oxidation is usually negligible in the\ndeveloping brain, this data provides novel evidence that is not the case following neonatal injury,\nwhere beta-oxidation is significantly upregulated. This metabolic shift results in a decreased\nVLCFA content in the brain, and interestingly, impacts cognitive performance (at P30) – in which\nsparing FA from oxidation can improve neurologic outcomes (the opposite to what is observed in\nadulthood, in which increased oxidation is associated with improved cognitive performance).\nImportantly, this paper demonstrates the unique specificity of the injured brain with regards to\nsubstrates utilized for oxidative metabolism during development, and results are promising for\nbeginning to investigate effective treatments following neonatal brain injury.\nSpecific comments:\nMajor comments:\n1. Palmitic acid is the primary product of de novo lipogenesis (DNL) and happens to be identified\nin males at P30 as the top differentially expressed FA in your volcano plot (Fig 1H; p < 0.001)\nbetween IHx and Nx groups, which is consistent with IHx males having a significantly higher\nhippocampal PAM at P30 than Nx males. Could DNL be increasing in your IHx group in\nattempt to maintain brain energy requirements and/or FA balance in PL in response to injury?\nEspecially since FASN (the major governor of DNL) was also upregulated at P30 in IHx males?\nNot a lot of discussion/hypotheses around this result, which also appears to be sex-specific.\nWe thank the reviewer for this comment and agree that increase in palmitic acid and de novo\nlipogenesis can be an endogenous attempt to recover. Our data on hippocampal de novo\nlipogenesis using acetate incorporation into lipids (Supplementary Figure 1) show no differences\nbetween Nx and IHx at all ages. We chose this assay because it quantifies acetate incorporation\nin both fatty acids and cholesterol while measured levels of palmitic acid reflect de novo\nlipogenesis from glucose as demonstrated by Smith et al. (2024) (PMID: 38233416)35.\nThis current revised manuscript now includes P22 total lipid fatty acid data, which also shows an\nincrease in palmitic acid after IHx (albeit only in males). This age (P22) was evaluated because\nmice are weaned at P21 and transition to a primarily carbohydrate rich rodent chow diet. These\nresults allowed us to delineate the trajectory of palmitic acid in the brain following IHx. In males,\nimmediately after IHx (P11), and during early recovery (P17), the levels of palmitic acid are\ncomparable to the levels observed in Nx hippocampus. However, at P22 and P30, the levels of\npalmitic acid significantly increase in the IHx group hippocampus indicating increase de novo\nlipogenesis. In contrast, females displayed palmitic acid levels equivalent to Nx hippocampus at\nall ages. Taken together, these data suggests that the increase in hippocampal palmitic acid may\nbe related to the recovery process that the brain is undergoing.\nWhile reviewing our data, we notice that at P22 and P30, there was more variability for C16:0 in\nfemales compare to males:\n% of total\nMean +/- standard deviation\nfatty Age Sex\nacids\nNx IHx\nMale 25.76 ± 0.265 26.23 ± 0.320\nP22\nFemale 25.27 ± 0.330 26.08 ± 1.248\n% C16:0\nMale 24.17 ± 0.904 25.31 ± 0.825\nP30\nFemale 23.51 ± 1.254 24.52 ± 2.489\nThe observed sex differences in brain lipid composition corroborate the results from the study by\nRodriguez-Navas et al. (2016) (PMID: 27656405)36 who showed sexual dimorphism in brain lipids\nof adult mice fed a low and high fat diet. Further studies focused on sex differences at different\nages are needed since there exists several possibilities that could explain these observation.\nRodriguez-Navas et al. (2016) suggested that the sex differences may be explained by\ndifferences in fatty acid uptake into the brain and expression of enzymes involved in fatty acid\nmetabolism, slower turnover of fatty acids, and possibly hormonal changes since they performed\nstudies using adult mice. Sexual dimorphism in mitochondrial respiration and oxidative stress has\nbeen reported in other models of neonatal brain injury (PMID: 27197831)37. Together with our\nresults, these data suggest that sex differences in brain metabolism and lipid composition are\nobserved prior to major changes in hormones associated with growth and maturation. These\npoints are now included in our discussion section.\n2. Almost all results shown are limited to the hippocampus, and not the whole brain (other than\nthe MS imaging data). How might you predict your findings to change at the level of the whole\nbrain, or other regions? If hippocampus was important a priori this could be mentioned.\nThe hippocampus was selected because it is important for learning and memory formation.\nImportantly, school aged children born extremely preterm display decreased hippocampal volume\nas well as cognitive deficits, specifically – learning and memory. We now expanded on the rational\nfor selecting the hippocampus in the introduction and discussion and we mentioned in the\nmethods that this brain region was selected a priori.\nBased on the MALDI-MSI imaging data at P17, we expect to have changes in other brain regions.\nCurrent and future studies aim to delineate changes in other regions (white matter, cerebellum,\nand cortex).\n3. Authors state there is a “reduction” in brain lipids in the abstract and elsewhere, however, total\nlipids in the hippocampus (primary region examined) are unchanged between IHx and Nx groups\n(% of total lipids per mg of tissue). Rather, it is lipid composition which changed. Language needs\nto be 1) altered to reflect this is not a real “reduction”, and 2) clarify this result is region-specific.\nWe agree with the reviewer and changes in the language were made. Specifically, in the abstract\nwe changed “reduction in fatty acids” to “change in hippocampal fatty acid profile”.\n4. Presenting hippocampal lipid composition data in a supplemental table – as both concentration\nand relative %, would be useful to the reader.\nThe reviewer highlights an important request to provide access to the lipidomic data. We agree\nthe lipid data will be made available. We will add the lipid data, both abundances and normalized\nvalues, to the supplementary information (Supplementary Tables 1,4,12, and 13) and source data\nas tables. We will also post the data to a publicly accessible database.\nThe reviewer mentions the supplemental data should include concentrations. We would like to\nclarify and more specifically describe how the lipid data was acquired and why the lipid data is\nnot reported as concentrations. The lipidomic data presented herein was acquired using a high-\nthroughput, targeted lipid profiling method. We have published previously using this methodology\n(e.g., PMID: 39128713, 38986724, 38057386)38–40. The data was acquired using a standard and\naccepted lipidomic method whereby lipid abundances were normalized to sum composition,\nmean-centered, and log-transformed. The data as presented does not represent concentration\nbut rather abundance. This allows for visualizing hundreds of detected lipids with multivariate and\nunivariate statistical analyses. In addition, the data was acquired using spiked-in internal\nstandards (Avanti Polar Lipids EquiSplash #330731) which provided one deuterium stable-\nlabeled lipid per lipid class. The lipid standards allow for compensation during lipid extraction,\nionization efficiency, and provide a means for determining relative abundance.\n5. There is an overall lack of justification surrounding why the hippocampus was selected for\nanalyses (see comment above), besides the obvious reason it corresponds to behavioral\nanalyses conducted. Is the hippocampus equipped for oxidation compared to others? Lipid\nsynthesis? This could be explained better.\nWe thank the reviewer for pointing this out. As mentioned above (comment #2) we selected the\nhippocampus because of its role in learning and memory and susceptibility to injury in children\nborn extremely preterm term. The additional benefit of focusing of the hippocampus is that it\nallows us to test function such as recognition memory and determine whether treatment (i.e.\nglycerol-triacetate) will improve the memory deficits.\nThe hippocampus is a highly energetically demanding region of the brain. We previously showed\nthat the hippocampus displays a high expression of the carnitine shuttle – required for long-chain\nfatty acid oxidation - compared to cortex and midbrain and has a high capacity to oxidize fatty\nacids (PMID: 28512781)41. We expanded our discussion to highlight these features.\n6. Pups were removed from the dam for extended periods of time very early in development to\nundergo cycles of hypoxia (16 x 3 min hypoxia + 5 min re-oxygenation x 2 = 32 cycles). This is a\nsignificant amount of time, and enough to impact maternal stress. Did authors measure and/or\nconsider maternal stress, and if this can program pups postnatally?\nWe did not consider maternal stress given that normoxia and intermittent hypoxia pups were\ntreated the same way. Both groups were separated from the mom for 16 cycles, return with the\ndam for 2 hours in the home cage, and separated again for the remaining 16 cycles. Normoxia\npups were kept in a separate chamber under normal oxygen levels for the 2x 16 cycles. We did\nnot observe malnutrition (for example, plasma carnitine levels) or maternal neglect in either of the\ngroups.\nMany models of neonatal brain injury require pups to be separated from the dam for a period of\ntime (ex: Hypoxia-Ischemia model) that can be as long as 90 minutes along with surgical\nmanipulations. In our model, there is limited handling of the dam therefore limiting stress.\n7. This is a lot of assays for a very small brain tissue to undergo. A supplemental table outlining\nwhich pups underwent which assay would aid in the reader understanding the results better.\nFurthermore, the use of “mice” throughout the methods should specify the age, and sex of the\nmice as authors used many timepoints and both sexes.\nWe thank the reviewer for the comment, and we would like to clarify that, due to a very small\ntissue amounts, we used a new set of mice for every experiment presented in this manuscript (i.e.\nFAO, acylcarnitines, lipidomics, total lipid fatty acids, MALDI, proteomics, RNA, etc.) All figure\nlegends report number of animals and sex used for each particular experiment. In addition, we\nprovided the exact number of mice used for each experiment as well as their age and sex in the\nsource data file.\n8. Were any other tissues collected from these mice? It would be nice to see the FA composition\nof a peripheral tissue like the liver and rule out if total lipids changed in peripheral tissues, but not\nthe brain. Perhaps there is between-tissue FA exchange to maintain total brain FAs.\nSince the brain is essentially dependent on continuous blood supply of substrates and precursors,\nwe extensively measured the blood levels of glucose, lactate, βHB, NEFA, and acylcarnitines.\nRecent data from Smith et al. (2024) (PMID: 38233416)35 showed that while peripheral tissues\n(liver) undergo remodeling, brain levels were maintained. In our data, NEFA were changed only\nat P22 – one day post-weaning from the dam. Plasma βHB levels were higher after IHx only at\nP11 and P17. This increased in βHB suggests that hepatic ketogenesis was upregulated up to\nP17. While peripheral tissue reprogramming after IHx would be an interesting focus for a future\nstudy, in this work, we aimed to delineate metabolic reprogramming in the hippocampus after IHx.\n9. IHx group weighs significantly less than Nx at most timepoints in both sexes. I understand this\nis necessary to model extremely preterm infants, however, this could also impact overall\nmetabolism, and substrates used for energy. Are hippocampal lipid alterations a result of hypoxia,\nweight loss, or both?\nThank you for your thoughtful comment. As this reviewer pointed out, we observed reduced body\nweight gain in the IHx group up to P30 in both sexes. As mentioned above (comment #8), the\nblood levels of substrates were maintained (glucose, lactate) or even increased (βHB) indicating\nthat delivery of substrates for brain energy and metabolism was comparable to Nx controls.\nTherefore, these results suggest that hippocampal lipid alterations were not due to a limited\nsupply of substrates for brain energy and growth.\nWhile substrate delivery was comparable to Nx, the fate of the substrates differ in the IHx group.\nIn this study, using biochemical and genetic approaches, we demonstrated that following IHx,\nastrocytes extensively oxidized long-chain fatty acids – a process that is dismal in the developing\nbrain – and affected subsequent hippocampal lipid composition.\nMinor comments:\n1. Abstract: The abstract overall is a bit boilerplate for the results described. Adding more detail\nto the results with regards to timepoints, sex, etc. would better capture your findings.\nThank you for this thoughtful comment. In this current version, we edited the text of the abstract,\nhowever, due to word limitation (150 words), only the most important results were provided.\n2. Abstract: Line 109: “timely” supply – again, we know this is a critical period of brain development\nbut what exactly are the timepoints that the intervention (triacetin) would have the most benefit?\nIn a separate manuscript that is currently under review, we determined that triacetate treatment\nafter neonatal brain injury from hypoxia was most efficacious when administered from P11 to P17\nand not at later timepoints. We also tailored our therapy to when fatty acid oxidation was occurring\n(P11 and P17 but not P22). We appreciate the reviewer’s comment and we removed the word\n“timely” from the abstract and introduction.\n3. Introduction: line 143: provide a citation for IHx as clinically relevant model of neonatal brain\ninjury?\nWe now included the appropriate citations:\nPMID: 254764923\nPMID: 2456983042\n4. Introduction: line 147: “clinically feasible source of acetate” – provide more detail supporting\nwhy this is clinically feasible? I assume it has a bitter taste; it is recognized safe as a food additive\nbut what dose would it need to be administered as, and would this be GRAS?\nThank you for this comment. We would like to further clarify that the US Food and Drug\nAdministration (FDA) classifies glycerol-triacetate as GRAS (https://www.ecfr.gov/current/title-\n21/chapter-I/subchapter-B/part-184/subpart-B/section-184.1901).\nClinically and in in vivo animal models, acetate cannot be administered on its own; it has to be\ndelivered as either sodium or potassium acetate and is usually administered intravenously in\nhumans. The concern is that in very vulnerable extremely preterm infants, the sodium or\npotassium may cause a significant metabolic and electrolyte disturbance. Glycerol-triacetate\nallows oral administration, is readily absorbed in the gut, and is an FDA compound not associated\nwith toxicity.\nIn our study, glycerol-triacetin (Millipore Sigma; 1030001000) was administered via gavage to\nensure that the full dose was given. At the dose that we administered (5mg/g body weight) we did\nnot see side effects. In order to determine the dose in humans, an IRB approved clinical trial\nwould be necessary. To bypass the bitter taste that can be associated with glycerol-triacetate,\nthe compound can be administered by oral gavage, which is often done in the neonatal intensive care\nunits (NICU).\nIn a study of two patients with Canavan disease, administration of glycerol-triacetate at 4.5g/kg\nwas well tolerated (PMID: 21474353)43.\nReference:\nPMID: 1600246144\n5. Language around timepoints selected and interpretation:\na. Line 153-154: the “neonatal period” authors selected not only impacts what would be a\npremature infant (PND 1-2/3 in a mouse), but also the window of a term infant (PND 7-10\nmouse) – translationally, are children that are born extremely preterm exposed to intermittent\nhypoxia as a term infant, as well?\nYes, children born extremely preterm that have apnea of prematurity persist until term or shortly\nthereafter. It is well established that nearly 100% of infants born EPT present with apnea of\nprematurity and the earlier the gestational age that they are born, the longer the apnea of\nprematurity persists.\nPMID: 928270545\nb. By P30 in a mouse, the brain is 90-95% of adult weight and corresponds to what would be a 2-\n3 year old child. Be careful with language around this timepoint referring to “later” in life.\nThank you for your comment. As mentioned, our response to reviewer #1 comment #1, rodent\nP30 corresponds to approximately 4-11 year old children. We change the language to avoid\nconfusion.\n6. Figure 1B-D and Extended Figure 1A-B: Add units for x-axis (i.e. “day 30” or “P30”). It is unclear\nwhat units you are referring to.\nThank you, the units for the x-axis have been corrected to P30 for Figures 1b-d and\nSupplementary Figure 1a, b, and e (Previously Extended Figure 1B-D).\n7. Figure 1E-F: Inconsistency in naming of PC 1/2 – pick either PC or component.\nWe used MetaboAnalyst to perform the PCA and PLS-DA analysis and generate the plots. Thus,\nwe kept the same labeling that was assigned by the software.\n8. I would consider adding the differentially expressed fatty acid data for females into the figure\nwith males, as it is equally as important and tells the whole story.\nWe thank the reviewer for the suggestion and agree. We created a new figure (Figure 2) that\nincludes both males and females total lipid fatty acid analysis.\n9. Extended data Figure 1C – are IHx males resistant to changes in body weight at 17 days? Or\nis this driven by variability, etc.?\nIt is driven by variability.\n10. Extended Figure 1C,D and elsewhere: Please clarify “age” on x-axis as number of days.\nWe edited the figures and labeled the x-axis of graphs across the manuscript as “Age (Days)”.\n11. Results: line 169-170: the primary substrate in humans and in rodents for DNL is glucose, not\nother fatty acids (they do indeed contribute but not to a great extent). Work has demonstrated\nthis, in the brain, during development: (I see you cite this already later on as well – it should be\nmentioned here for consistency).\nThis citation has been added.\n12. Results: Line 180-182: “prolonged dysregulations in lipid homeostasis” – what kind of\ndysregulations?\nIncreased/decreased DNL? Increased/decreased oxidation? A bit of an oversimplified statement for your detailed results.\nThank you for the comment. We described the lipid profile changes between the two groups in\nthe paragraph where we write that specific closing sentence. The extent of dysregulation is now\ndiscussed in detail in the discussion section.\n13. The concept of astrocytes being specialized to oxidize fatty acids is well-established (for\nexample: (https://onlinelibrary.wiley.com/doi/10.1111/gtc.13144) – is your finding novel in the\ntimepoints/model selected? What makes your finding novel?\nWhile the concepts of astrocytes being able to oxidized fatty acids has been known for decades\n(PMID: 13576157, PMID: 2002348)46,47, this phenomenon is aged-dependent and is negligible in\nthe developing brain. To date, fatty acid oxidation was examined in adult mice and in a model of\nAlzheimer disease (PMID: 37460843, PMID: 36959514)48,49.\nThe novelty of our study is that this is the first study to report: 1) fatty acid oxidation occurs\nspecifically in the developing brain following a model of neonatal brain injury due to prematurity,\n2) fatty acid oxidation was still present 6 days after the injury during the recovery period under\nnormoxic conditions, and 3) in vivo administration of glycerol-triacetate significantly reduce\nhippocampal fatty acid oxidation by ~71% in the IHx group.\n14. Discussion is somewhat brief – more comparisons to the existing literature could be made on\nthis topic.\nThank you, we extensively revised and expanded the discussion of our findings.\n15. What kind of diets were the pups on post-weaning, or the dams throughout the study? This is\nnot mentioned in the manuscript.\nDams and weaned pups were fed a standard rodent Chow diet. We now specified this in the\nmethods section.\n16. Were the mice bred in-house, or were the dams ordered pregnant? This is not clear.\nDams were bred in-house. To ensure variability and limit in-breeding, each dam was used only\ntwice and each time with a new unrelated male. In the methods subsection “Animals”, we edited\nthe text from “The colonies were maintained in the animal facility…” to instead say “Mice were\nbred in-house and the colonies maintained in the animal facility…” for clarification.\n17. What methods were used for randomization? (line 405, line 419)\nDams were randomized using simple randomization when pups were P1 to undergo Nx or IHx.\nPups from different litters were used for each experiment and timepoint.\n18. Who conducted the behavioural tests, as well as most analyses? Were investigators blinded\nor unblinded to treatment?\nThe investigator who performed the behavioral tests was blinded to the treatment groups. RFF\nwho performed the treatment with glycerol-triacetate and analyzed the data was not blinded. A\nstatement was added to the “Behavior” subsection within the Methods section for clarification.\n19. Clarify what portion of the brain was used for lipidomics (line 516) – was this from the same\nbrains that the hippocampus was taken from? Left vs. right hemisphere? Remaining whole brain?\nWe apologize for the lack of clarity. The lipidomics was performed on microdissected\nhippocampus (combined right and left) to ensure adequate tissue amounts for lipidomics analysis\nspecifically at a younger age. We corrected the methods from “brain” to “hippocampus”.\n20. Sample size is listed as a range in figure legends (ex. 7-13/sex/group/treatment) – as per\nNature Comm requirements, sample size must specify the total number of mice per sex, per\ntreatment and not be presented in a range format.\nDue to the multiple number of groups (age/sex/treatment) in each experiment, we listed the range\nin the figure legend. However, we specify the exact number in the Source Data file for every\nexperiment.\n21. Sample size for NOR test is rather small, is this enough power to detect changes?\nWe performed the behavioral test on P30 Nx and IHx male (n=8/group) and female (Nx, n=5; IHx,\nn=9) mice. Since there were no differences between the sexes, both males and females in the\nIHx had worse performance in comparison to the Nx group, we combined mice of both sexes with\na total of 13-17 mice per group.\n22. Were the RT PCR data validated?\nIn this work, we did not perform RNA-seq. We performed target RT-PCR of specific genes of\ninterest across all the ages/sexes/groups studied. Primers sequences were obtained from\npublications or from the Harvard Primer Bank.\n23. Authors refer to VLCFA as FA > 20 carbons – is it 20 or 22 that deems them VLCFA? Check\nIUPAC\nThank you for this helpful comment, this is now specified in the text as “≥ 20 carbons or more”.\n22 carbons will be considered VLFCA in the text.\n24. Lines 187-189, you state the developing brain uses fatty acids for brain lipid synthesis, then\nfollow it with the developing brain extensively using glucose for de novo synthesis of fatty acids –\nthese statements contradict each other.\nThank you for the comment. We edited the manuscript for clarification to “….while fatty acids\nimported from the circulation are used for the synthesis of complex lipids in the brain”.\n25. As per Nature Comm requirements “as previously described” must be replaced with exactly\nhow the procedure was carried out (line 661) in the methods sections\nThank you for the thorough review. This is now detailed in the methods section.\n26. Blood measurements in pups – these were unfasted or fasted?\nBlood from P11 mice was collected immediately after the last cycle of IHx (~ 2hrs away from the\ndam). At all other ages (P17, P22, and P30), mice were not fasted. Both Nx and IHx mice were\ntreated the same. We clarified in the figure legend (Supplementary Fig. 3) that the data were\nobtained from fed-state mice.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #5 (Remarks to the Author):\nThe manuscript by Fernandez et al. provides new evidence that during the critical period of brain\ndevelopment in neonates born extremely prematurely, brain mitochondria increasingly rely on\nfatty acid oxidation. This metabolic shift coincides with a reduction in total brain fatty acids and\ncomplex lipid classes, which may contribute to behavioral changes. The authors demonstrate that\nsupplementation with alternative fuels, such as acetate, in in vivo and in vitro models, spares fatty\nacids and leads to improved neurological outcomes. Authors use of intermittent hypoxia as a\nmodel to study neonatal brain injury in extremely preterm infants. Overall, the manuscript is well\npresented, and the major conclusions are supported by the data.\nThe reviewer suggests the following improvements to enhance the clarity and rigor of the\nmanuscript:\n1. Authors need to improve the consistency in Definitions of Fatty Acid Classes. In line 355, the\nauthors consider very long-chain fatty acids (VLCFA) as lipids with more than 22 carbons;\nhowever, in Figures 1J/K (and similarly in Extended Figures 2C/D, 9B/C), the cutoff appears to\nbe 20 carbons. Can the authors reconcile this discrepancy between the results and conclusions?\nFor consistency, it is generally accepted that long-chain fatty acids (LCFA) include lipids with 12–\n20 carbons, while VLCFA are those with 22–38 carbons. Distinguishing between these two\ncategories is important, as the data treat all fatty acids similarly, yet the discussion focuses\nprimarily on VLCFA.\nWe thank the reviewer for the pointing out this discrepancy. In this revised version of the\nmanuscript, we corrected both results and discussion sections.\n2. Presentation of Lipid Remodeling Data could be also improved. The changes shown in lipid\nremodeling of free fatty acids appear modest (Figures 1I, Extended Figures 2B, 4F, 5D, 3C, and\n9A), and the graphs make it difficult to discern statistically significant trends. Rather than\ndisplaying all data, including non-significant changes, the authors could provide a comprehensive\ntable summarizing all results and only depict statistically significant changes in the figures for\nclarity.\nIn this submission, we provide raw data containing total lipid fatty acids and acylcarnitines in excel\nformat as well as all statistical analyses (Source data and Supplementary Information). However,\nto reconcile the critiques from reviewers 3 and 5, we are displaying the results as heatmaps in\nthe main figure and report percent of fatty acids from total fatty acids in a supplemental table for\nall ages and sexes.\n3. Could the observed phenotype be explained simply by low body weight and, consequently, a\nreduced capacity to accumulate adipose tissue, which is essential for normal brain development?\nThe authors are encouraged to address this possibility.\nWe thank the reviewer for this interesting and intriguing point. The brain relies on a constant\nsupply of substrates for growth, development, and metabolism. This supply is fulfilled by delivery\nof these substrates via systemic circulation. Our results show that IHx did not result in\nhypoglycemia and did not decrease blood levels of lactate, βHB, and NEFA. At P17, 6 days post-\ninjury, we continue to observe in the IHx group increased blood levels of βHB generated in the\nliver and comparable levels of NEFA to Nx. Indeed, recent elegant studies by Bazinet’s group,\nshowed that glucose is the primary substrate for de novo lipogenesis in the developing brain\n(PMID: 38233416)35. Furthermore, this group demonstrated that mice fed a low palmitic acid\n(C16:0 – primary product of de novo lipogenesis) diet, preserved brain palmitic acid levels by\nupregulating hepatic synthesis from dietary sugars. We want to point out that neither levels of\nbrain palmitic acid or essential fatty acids (ω-6 and ω-3) were decreased following IHx at any age\nstudied. Instead, we observed decreased levels of ≥20 carbon-long saturated and ω-9 fatty acids,\nwhich are synthesize locally (in the brain). Taken together, these results suggest that there was\nno compromise in nutrition or circulating levels of substrates for de novo lipogenesis, and the\nobserved changes are due to disrupted lipid metabolism in the brain.\nClinical observational studies extensively report that newborns born preterm have low birth weight\nand are closely monitored for weight gain and growth during subsequent NICU stay. By the age\nequivalent to full term newborn (40 weeks), these infants have total adiposity comparable to term\nnewborns (PMID: 15611357, PMID: 10212079)50,51. Most (~80%) of preterm infants experience\ncatch up in growth by age 2-3 years, while in the other 20%, this catch up growth can last until\nadolescence (PMID: 18953169, PMID: 11044763, PMID: 15795505, PMID: 16291360, PMID:\n15033844)52–56. Adults born preterm with very low birth weight, display shorter height and\nequivalent adipose tissue compared to those born full term (PMID: 37973945)57. Indeed, our\nresults show that we recapitulate in mice the clinical observed phenomenon of low body weight\nthroughout development. Ongoing studies in our lab are further characterizing body fat, lean\nmass, and adiposity in our model of IHx using EchoMRI 100 QNMR instrument.\n4. The authors report that supplementation with glycerol-triacetate improves neurological\nrecovery based on NOR scores. Does this treatment also result in improvements in brain lipidomic\nprofiles, particularly in the hippocampus? Including this data and more presenting more\nphenotypic data would strengthen the conclusions.\nWe thank the reviewer for the suggestion. This revised submission now includes the new results\nregarding the effects of glycerol-triacetate on brain lipid composition. Specifically, we report body\nand brain weight, hippocampal percent of total lipid per mg of hippocampus, and total\nhippocampal lipid fatty acid analysis.\nOur results show that treatment with glycerol-triacetate after IHx restored hippocampal total lipid\nfatty acid composition. Specifically, the levels of ≥20 carbon-long saturated and ω-9 fatty acids\ndecreased in the IHx-injured mice group were comparable to the normoxia controls following\ntreatment with glycerol-triacetate. These data are now reported in figure 7 and supplementary\nfigure 9.\nMinor Suggestions:\n- The term “differentially expressed lipids” is somewhat misleading. Lipids, unlike genes, may\nnot “express” information or functions in the traditional sense; many serve structural roles, such\nas membrane components. The authors are encouraged to clarify or replace this terminology.\nThank you. We changed the title of the heatmap and the text to “Significantly changed lipids”.\n- Extended Figure 1C-D: It would be valuable to include pre-intervention data as a control to\nconfirm that both groups started with comparable body weights.\nThe mice we use are C57BL6/J mice without genetic interventions. We collected the body weight\nat P1 before IHx and included it in the graph (Supplementary fig. 1c, d). However, in compliance\nwith the NIH three “R” (replacement, reduction, refinement), we did not collect brain weights at\nP1 prior to IHx.\n- Extended Figure 4: The data likely represent fed-state levels; the authors should clarify this in\nthe figure caption.\nThank you for the suggestion. As per this reviewer’s request and Reviewer #3 comment 26, the\nfigure legend was updated to “Data for each metabolite are presented for fed-state mice and\nas combined sexes, male only, and female only.”\nREFERENCES:\n1. Poets, C. F. et al. 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Jussinniemi, L. et al. Body composition in adults born preterm with very low birth weight.\nPediatr Res 95, 1316–1324 (2024).\nResponse to reviewers:\nWe thank all three reviewers for their expedited review of this resubmission. All reviewers had no\nfurther comments or suggestions."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have appropriately addressed our concerns and there are no further comments for the revision.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors have done a really nice job at addressing our previous concerns.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "The authors have made detailed revisions in response to the reviewers' comments. The manuscript is therefore recommended for acceptance."}, "author_response": ""}]

Decision letter