ABSTRACT BACKGROUND Neonatal encephalopathy (NE) remains a common cause of infant morbidity and mortality. Neuropathological corollaries of NE associated with acute hypoxia-ischemia include

a central injury pattern involving the basal ganglia and thalamus, which may interfere with thermoregulatory circuits. Spontaneous hypothermia (SH) occurs in both preclinical models and

clinical hypoxic-ischemic NE and may provide an early biomarker of injury severity. To determine whether SH predicts the degree of injury in a ferret model of hypoxic-ischemic NE, we

investigated whether rectal temperature (RT) 1 h after insult correlated with long-term outcomes. METHODS Postnatal day (P)17 ferrets were presensitized with _Escherichia coli_

MRI, and neuropathology were quantitated. Whole-brain volume and fractional anisotropy in SH brains were significantly decreased compared to control and NT animals. SH brains also had

significantly altered gyrification, greater cortical pathology, and increased corpus callosum GFAP staining relative to NT and control brains. CONCLUSION In near-term-equivalent ferrets,

nesting RT 1 h after HIH may predict long-term neuropathological outcomes. IMPACT * High-throughput methods to determine injury severity prior to treatment in animal studies of neonatal

brain injury are lacking. * In a gyrified animal model of neonatal inflammation-sensitized hypoxic-ischemic brain injury in the ferret, rectal temperature 1 h after hypoxia predicts animals

who will have increased cortical pathology and white matter changes on MRI. * These changes parallel similar responses in rodents and humans but have not previously been correlated with

long-term neuropathological outcomes in gyrified animal models. * Endogenous thermoregulatory responses to injury may provide a translational marker of injury severity to help stratify

animals to treatment groups or predict outcome in preclinical studies. You have full access to this article via your institution. Download PDF SIMILAR CONTENT BEING VIEWED BY OTHERS

HYPOXIA–ISCHEMIA-MEDIATED EFFECTS ON NEURODEVELOPMENTALLY REGULATED COLD-SHOCK PROTEINS IN NEONATAL MICE UNDER STRICT TEMPERATURE CONTROL Article 19 February 2022 BLOOD GLUCOSE AND

Β-HYDROXYBUTYRATE PREDICT SIGNIFICANT BRAIN INJURY AFTER HYPOXIA-ISCHEMIA IN NEONATAL MICE Article 24 August 2024 SLOW REWARMING AFTER HYPOTHERMIA DOES NOT AMELIORATE WHITE MATTER INJURY

AFTER HYPOXIA-ISCHEMIA IN NEAR-TERM FETAL SHEEP Article Open access 05 August 2024 INTRODUCTION Perinatal hypoxia-ischemia (HI), and subsequent neonatal encephalopathy (NE), is the third

most common cause of neonatal mortality globally.1 Therapeutic hypothermia (TH) is the current standard of care to mitigate consequences of moderate or severe hypoxic-ischemic encephalopathy

(HIE, i.e., NE associated with acute HI in term and near-term infants) as well as in animal models of hypoxic-ischemic NE.2,3,4 Although TH has been successful in reducing the occurrence of

poor outcomes from HIE, it is currently only indicated in high resource settings,3 and a significant proportion of surviving infants still develop long-term disabilities; thus, the search

for supplementary therapies is ongoing.5,6,7 Early biomarkers of brain injury are needed to accurately select those infants who will benefit from TH, as well as to predict long-term

outcomes. Such biomarkers are also needed for both clinical and preclinical studies as treatment optimization may require stratification of different injury severities to different

therapeutic strategies.8 Previous animal models of hypoxic-ischemic NE have utilized neuroimaging techniques like magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) to

identify biomarkers of brain injury, but these methods are expensive and difficult to implement at the scale needed in robust animal studies.9,10 Spontaneous hypothermia (SH) after acute

brain injury has been associated with injury severity,11,12 and recent observational studies found that human infants with higher injury severity and worse long-term outcomes required less

active cooling to attain target cooling temperature during TH, presumably due to injury-associated SH.13,14,15 While all brain regions are susceptible to injury, more severe insults often

result in basal ganglia and thalamus damage.16 This injury pattern may involve nearby thermoregulatory circuits,17,18 which would explain SH observed in clinical HIE19 and support the link

between HIE and disruption of the thermoregulatory circuitry.11,12 Performing adequately powered, rigorous, and reproducible preclinical studies to explore potential therapies for

hypoxic-ischemic NE that are not cost prohibitive requires easily obtainable, non-destructive metrics for use as biomarkers of injury.20 Here, post-insult rectal temperature (RT) data in an

inflammation-sensitized hypoxia-ischemia/hyperoxia (HIH) ferret model of NE were reviewed to determine whether early spontaneous core temperature changes following exposure to HIH are

associated with long-term neuropathological outcomes. We have previously shown that the ferret is an excellent mid-sized animal model in which to study the long-term impacts of

hypoxic-ischemic NE.21,22 We hypothesized that ferret kits with SH following HIH would have greater injury compared to HIH littermates who remained normothermic (NT) after the insult.

METHODS AND MATERIALS ANIMALS AND HOUSING The Institutional Animal Care and Use Committee at the University of Washington approved all animal experiments. The primary outcome data from these

same animals were previously published.21 P17 INJURY MODEL At P17, which is near-term equivalent, ferret kits were weighed, and randomized to control or HIH groups. Animals in the HIH

groups received 3 mg/kg lipopolysaccharide (LPS, Ultrapure from _Escherichia coli_ 055:B5, List Biological, CA) intraperitoneally 30 min prior to a bilateral carotid artery (CA) ligation

where the left CA was permanently ligated twice with silk suture (5–0, Fine Science Tools, Foster City, CA) and the right CA (RCA) was temporary occluded with umbilical tape. Following

surgery, kits in the HIH groups were exposed to 2 h of hypoxia-hyperoxia-hypoxia (9%, 80%, 9%). The RCA occlusion was removed after the second round of hypoxia, as previously described.21

Control animals received 3 mL/kg saline and were separated from their jill and monitored in the surgical suite for the duration of the surgeries. Following the HIH insult, all ferret kits

were placed back in the nest for 1 h to feed and maintain hydration. Animals were subsequently removed from the nest and immediately had their RT gauged to establish post-surgical nesting

temperature (Precision 4000A thermometer, YSI, Yellow Springs, OH). HIH animals were randomized to saline Vehicle (Veh, _n_ = 25), Erythropoietin (Epo, _n_ = 21) (2000 IU/kg s.c. at 0, 24,

48 h, and day 7 for 4 total doses) or TH (33.5 °C for 6 h, _n_ = 24) treatment groups, as previously described.21 Nesting RT was measured prior to administration of any therapies, with

assigned treatments initiated 90–120 min after HIH. All animals were either placed in a plastic chamber in a water bath adjusted to maintain normothermia (target RT of 36.5 °C) or placed in

a separate, cooled water bath to receive TH (target RT 33.5 °C) for 6 h. During temperature maintenance, RT was determined in every animal every 15–30 min. After 6 h of temperature

management, all animals were returned to their nests. A flow diagram of injury and temperature management is shown in Fig. 1. ESTABLISHMENT OF A P17 FERRET NESTING TEMPERATURE NORMAL RANGE

Historical control P17 ferret kit data were used to establish a normal resting RT range (Fig. 2). Control ferret kits exhibited a mean (SD) nesting temperature of 35.5 °C (0.63 °C) and HIH

ferret kits exhibited a median post-surgical nesting temperature of 34.0 °C (1.05 °C). For statistical analyses, a “normal” post-surgical nesting temperature range was determined as mean ±2 

SD in healthy control animals (range 34.4–37.4 °C, _n_ = 23). Based on RTs taken 1 h after the insult, HIH-exposed animals were separated into NT (≥34.4 °C) and spontaneously hypothermic

(SH; <34.4 °C) groups. BRAIN MEASUREMENTS At P42, the kits were euthanized and perfusion-fixed with phosphate-buffered saline (PBS) and 10% neutral buffered formalin. After further

post-fixation in formalin, brains were removed and lightly dried with tissue paper to remove excess liquid. The external features and dimensions of the brain were measured using digital

calipers with fine pointed jaws (SRA Measurements Products, Walpole, MA), as previously described.21,22 Sulci were measured from the beginning and end of the most distinct portion of the

corresponding sulcus. Gyri were measured from the widest aspect of each corresponding gyrus. Bilateral measurements of the lateral, suprasylvian, coronal, pseudosylvian, ansinate, cruciate,

and presylvian sulci were summed and compared. The bilateral measurements of the lateral, suprasylvian, coronal, ectosylvian, orbital, and posterior sigmoid gyri were also measured. All

measurements were completed by a researcher that was blinded to treatment groups. Brains were subsequently returned to formalin for future ex vivo quantification. EX VIVO QUANTIFICATION

Methodology for ex vivo MRI, immunohistochemistry (IHC), and quantitative IHC (qIHC) have been previously published.21 For ex vivo MRI, brains were removed from formalin before being rinsed

and stored in PBS at 4 °C to rehydrate for 72 h. Agarose gel sleds were fixed within 50 mL Falcon tubes. Brains were then mounted on the agarose gel sleds and immersed in Fomblin

(Perfluoropolyether, PFPE; Solvay Specialty Polymers, GA) for MRI with DTI from which fractional anisotropy (FA) values were calculated. Volumetric analyses of the whole brain, cerebellum,

brainstem, cerebrum, and hypothalamus were conducted. Following brain measurements and MRI, coronal slices at the level of the caudate nucleus and thalamus were taken from the brains and

embedded in paraffin. Four µm sections were then taken for hematoxylin and eosin (H&E) and IHC staining, including glial fibrillary acidic protein (GFAP, 1:300 dilution, Agilent (Dako)),

Iba-1 (1:1500 dilution, WAKO Chemicals, 019-19741), myelin basic protein (MBP, 1:500 dilution, Abcam, AB7349), and oligodendrocyte transcription factor 2 (Olig2, 1:500 dilution, Millipore,

AB9610), as previously published.21 H&E-stained slides were evaluated by a board-certified veterinary pathologist who was blinded to experimental manipulation and were scored for

cortical lesion (0–4) and mineralization (0–4) on a total scale of 0–8. The IHC slides were scanned using a Nanozoomer Digital Pathology slide scanner (Hamamatsu; Bridgewater, NJ) with a 20×

objective. Regions of interest (ROIs) for quantitative analysis were manually traced on all IHC images using the Visiopharm software (Hoersholm, Denmark), which calculated ratios of

positively stained tissue to unstained tissue for each ROI. ROIs included the thalamus, subcortical white matter (SWM), dorsal cortex, hippocampus, and corpus callosum (CC). The ROIs were

then quantitatively assessed as a ratio of stained tissue to total tissue areas. GFAP-stained slides were also imported into NDP.view2 (Hamamatsu Photonics, Bridgewater, NJ) to assess the

thickness of white matter tracts within the sub-sulcal regions of the SWM. SWM tracts were measured at standardized regions inferomedial to the lateral sulci, suprasylvian sulci, and

pseudosylvian sulci by a blinded researcher using the NDP annotation tool. The images were analyzed at 0.63× and were gamma corrected (0.5) to accentuate the staining of the SWM tracts.

STATISTICAL ANALYSIS Descriptive analyses were used to describe nesting RT and weight gain by group. For comparison of outcomes, NT and SH animals were compared to uninjured littermate

controls using linear models with robust standard errors adjusting for the treatment group (Veh, Epo, or TH) using the rigr package in R. The SH group was then compared to NT using linear

contrast with the generalized linear hypothesis testing (glht) function. Statistical comparison of group FA values was performed by threshold-free cluster enhancement adjusted for multiple

comparisons, with significance levels set at 0.05 and 0.10. .All remaining statistical analyses were performed in R (Version 4.1.2, Foundation for Statistical Computing, Vienna, Austria).23

Figures were made using Prism version 9 (GraphPad software). _P_ values <0.05 were considered statistically significant. RESULTS TEMPERATURE OUTCOMES The NT group consisted of 27 animals

(TH: _n_ = 9, Epo: _n_ = 9, Veh: _n_ = 9) and the SH group consisted of 43 animals (TH: _n_ = 15, Epo: _n_ = 12, Veh: _n_ = 16). The sex balance of the HIH groups was similar—14 females and

13 males in the NT group compared to 23 females and 20 males SH in the group. The control group contained 11 female animals and 12 male animals. Seven animals (NT: _n_ = 1, SH: _n_ = 6) died

during or after the temperature management period after HIH. Prior to death, animals that died were significantly cooler than control animals (_p_ < 0.0001) and nonsignificantly cooler

than other animals that underwent HIH (_p_ = 0.06; Fig. 2). These animals were not included in subsequent analyses. MODEL OUTCOMES At P17, all animals had a median (IQR) weight of 87 g

(80–92.5 g; Fig. 3a). Following HIH, male and female animals both lost a median weight of 9 g (8–11 g) by P18. By comparison, control males gained a median weight of 12 g (11–16 g) and

control females gained a median weight of 9.5 g (7–11.8 g) by P18. After P18, all animals had a similar weight gain pattern. At P42, the control animals had a median weight of 322.5 g

(303–371.6 g; Fig. 3b). Compared to controls, NT animals were lighter with a median weight of 305 g (272–329 g), but this difference was not significant. SH animals had a median weight of

283.5 g (268–319 g), which was significantly decreased relative to controls (_p_ = 0.003; Fig. 3b). MRI BRAIN VOLUMES On MRI, SH brains had significantly decreased whole-brain volume

relative to control and NT brains (_p_ = 0.005 and _p_ = 0.001, respectively; Fig. 4a). Compared to controls, hypothalamic volumes were significantly decreased in SH brains (_p_ = 0.044;

Fig. 4b). NT brains had similar hypothalamic volumes compared to controls but did not significantly differ from SH hypothalamic volumes. SH hypothalami appeared to remain more medial in the

brain and were contained to fewer cross-sectional MRI images relative to NT and control hypothalami. SH brains also exhibited significantly decreased cerebellar volumes when compared to

controls (_p_ = 0.027; Fig. 4c). Full MRI brain volumetric data are described in Table 1. SH brains had a significantly higher ratio of cerebellar to whole-brain volume relative to both

control and NT brains, suggesting relative sparing of the cerebellum with injury (data not shown). SH brains had significantly decreased ratios of cerebrum to brainstem volume compared to

control and NT brains had significantly increased ratios of (_p_ = 0.0002 and 0.0014, respectively; Fig. 4d). MRI FRACTIONAL ANISOTROPY At a significance level of 0.05, SH brains had

significantly reduced FA unilaterally in the right posterior cerebral white matter tracts adjacent to the hippocampus compared to NT brains (Fig. 4e). At a significance level of 0.10, SH

brains had significantly reduced FA bilaterally throughout the anterior and posterior cerebral white matter compared to NT brains (Fig. 4f). There were no hypothalamic FA differences between

groups. BRAIN MEASUREMENTS Summed sulcal lengths were significantly decreased in the NT (_p_ = 0.0042) and SH (_p_ < 0.0001) brains compared to controls (Fig. 5a). Compared to NT brains,

SH brains exhibited significantly decreased summed sulcal lengths (_p_ = 0.0013). NT and SH brains had significantly shorter lateral sulci relative to controls (_p_ = 0.0061 and _p_ < 

0.0001, respectively; data not shown). SH brains had significantly shorter lateral sulci compared to NT brains (_p_ = 0.017). Like the lateral sulci, control brains had longer coronal sulci

lengths compared to both NT and SH brains (_p_ = 0.021 and _p_ < 0.0001, respectively; data not shown). SH brains had significantly decreased summed gyral widths relative to controls and

NT brains (_p_ = 0.0004, _p_ = 0.046; Fig. 5b). Control brains had significantly wider coronal gyri compared to SH brains (_p_ = 0.01; Fig. 5c) and there was no difference between NT and SH

brains. Full brain measurement data are described in Table 1. CORTICAL PATHOLOGY Control brains had a median (IQR) cortical pathology score of 0 (0–0). NT brains had a median cortical

pathology score of 0 (0–1), which was not significantly different compared to control brains. SH brains had a median cortical pathology score of 1 (0–3), which was significantly higher

relative to control brains (_p_ = 0.0024; Fig. 5d). Similarly, SH animals had significantly higher cortical pathology scores compared to NT animals (_p_ = 0.022). QIHC GFAP staining was

significantly increased globally in the SH brains compared to control brains (_p_ = 0.03; Fig. 6a). However, both NT and SH brains had higher ratios of GFAP-stained tissue area to total

tissue area in the thalamus (_p_ < 0.0001, _p_ = 0.001) and SWM (_p_ = 0.038, _p_ = 0.025) relative to control brains (data not shown). In the CC, SH brains had significantly increased

GFAP-stained tissue area to total tissue area compared to NT and control brains (_p_ = 0.03, _p_ = 0.003; Fig. 6b). Gross MBP staining relative to gross tissue area was significantly

increased in the SH brains when compared to control brains (_p_ = 0.041; Fig. 6c, e). NT brains also displayed decreased MBP staining relative to SH brains, but the difference was

nonsignificant. A similar but nonsignificant MBP staining outcome was observed in the CC of the SH brains (Fig. 6d). No significant differences were observed in the Olig2 and Iba-1-stained

ROIs across all groups (data not shown). In the white matter ROI inferomedial to the lateral gyri, the SH brains exhibited significantly narrower white matter tracts compared to controls

(_p_ = 0.014; not shown). There was no significant difference between NT and SH brains. In the white matter tracts inferomedial to the suprasylvian gyri, SH brains had significantly narrower

white matter tracts compared to both control and NT brains (_p_ = 0.006 and _p_ = 0.012, respectively; not shown). When the MBP-stained slides were used to measure CC width, the thickness

of the CC was significantly reduced in both NT and SH brains (_p_ = 0.004 and _p_ < 0.0001, respectively; not shown). However, NT brains displayed a nonsignificantly wider CC compared to

SH brains. In the white matter ROIs adjacent to the lateral, suprasylvian, cingulate, and pseudosylvian gyri, SH brains had significantly narrower white matter tracts across all summed ROIs

relative to the NT and control brains (_p_ = 0.036 and _p_ = 0.009, respectively; not shown). Full white matter tract measurement data are described in Table 1. DISCUSSION Using data from a

historical cohort of near-term equivalent ferrets that underwent a P17 HIH insult, we examined the relationship between spontaneous post-insult RT and injury severity. SH brains had

significantly decreased FA in the right posterior white matter tracts adjacent to the hippocampus compared to NT brains, which suggests that SH animals experienced more white matter

dysgenesis than NT animals.24 Ferrets that exhibit SH also demonstrated several signs of severe brain injury including higher cortical pathology scores, increased GFAP staining in the CC,

and increased density of MBP staining. This may be indicative of increased astroglial25 and oligodendroglial26 activity in SH animals, which can have both beneficial and deleterious

implications following brain injury. The SH animals also exhibited abnormal white matter tract development and decreased brain gyrification.21,22 These results suggest that post-HIH nesting

temperature is predictive of injury severity in a ferret model of hypoxic-ischemic NE and offers an easily obtainable metric to use in future studies that stratify or randomize treatment

modalities based on injury severity. In a previous developmentally equivalent P7 rat study of unilateral HI, RTs 1 h after injury were associated with global pathology scores one week after

injury.10 We used a similar methodology to determine associations between post-insult temperature and long-term outcomes using a gyrified ferret model of hypoxic-ischemic NE. Our analysis

showed that SH brains had significantly higher cortical pathology scores and greater injury-associated IHC outcomes compared to control and NT animals. This suggests that NT animals

experienced milder injuries relative to SH animals. As small animal models of neonatal HI display highly variable neuropathology with the same insult, our results support the possibility of

using post-HI temperature to stratify animals based on the severity of the initial injury. Similar associations between post-HI temperature dysregulation and short-term injury severity have

also been observed in clinical cases of hypoxic-ischemic NE. Burnard and Cross (1958) were the first to observe a spontaneous decrease in core temperature following perinatal asphyxia in

term infants.19 Since these initial observations, researchers have endeavored to understand the clinical relationship between temperature dysregulation and the HI insult.10,11,27 More

recently, lower core temperatures following HI in neonates with HIE have been correlated with varying unfavorable outcomes, including more severe encephalopathy and lower day 4 Thompson

scores.10,13,14 We have also previously shown that infants with HIE that experience favorable outcomes required about 4 °C more active cooling on a servo-controlled cooling mattress while

receiving TH over the course of 72 h compared to neonates with unfavorable outcomes.14 These results were strongly correlated with MRI injury and significantly correlated with deep gray

matter injury, suggesting that infants with more severe injury are SH and therefore require less active cooling. We and others have also shown that mattress temperatures are highly

predictive of long-term outcomes in infants with moderate-severe HIE.13,15 For instance, infants from the Optimizing Cooling trial treated with TH at 33.5 °C who required consistently low

mattress temperatures had 100% disability-free survival.15 Similarly, the post-HI temperature of ferret kits was strongly associated with MRI and white matter alterations in addition to

increased neuropathology. In particular, SH animals had narrower but denser white matter tracts, as well as significantly reduced FA, compared to NT and control animals. We hypothesize that

thin, highly MBP-stained white matter tracts with decreased Olig2 expression are the result of dysmature white matter tracts that are hypermyelinated, but disorganized relative to normal

development at that age. However, more work is required to determine how white matter structure is altered in the model in general and SH animals in particular, as well as the mechanisms by

which tract density increases in the setting of apparent decreased homogeneity. Previous clinical publications have suggested that post-HI temperature dysregulation could be associated with

damage to the thermoregulatory circuits located adjacent to the central deep gray matter.14 HI insults have also been shown to affect the hypothalamus, which lies directly inferior to the

central deep gray matter of the ferret brain.28 To address these hypotheses, we analyzed hypothalamic volumes from the ex vivo MRIs.29 Although SH brains had significantly reduced

hypothalamic volumes compared to NT and control brains, this significance was lost after adjusting for brain volume, suggesting that the smaller hypothalami may be due to smaller brains

overall as a result of globally disrupted brain development. In addition, no differences in FA within the hypothalami were noted across groups. However, during inspection of the hypothalami

on MRI, a subjective assessment suggested that some hypothalami in the SH group did not extend as far rostrally as observed in the control hypothalami. A more detailed assessment of

hypothalamic injury and development is necessary to understand the morphometric differences of the hypothalami in these animals and how it may impact the brain following HI. This study has

several limitations and potential confounding variables, including those due to the housing of the animals. The ferrets are housed in a large cage with a small plastic container acting as a

nest where the jill rests with the kits. Although the kits were all exposed equally to the jill during the rest periods, it is possible a small component of endogenous temperature regulation

could have been influenced by nest location. In our previous experience with rodent models of HI, the dam does not always cover the entire cohort and some pups are exposed to cooler cage

conditions, which could affect animal temperature management. Future studies could gather more comprehensive data on jill temperature and the temperature fluctuations of the nesting box. A

number of limitations are also related to the clinical correlates of the injury model and interpretation of the findings. The model used here is approximately equivalent to human infants of

32–36 weeks’ gestation and therefore may be considered “late preterm” or “near-term,” similar to the P7 rat.21,22 Moderate-severe HIE treated with active TH is currently only indicated for

infants of 36 weeks’ gestational age or older at birth, and though our findings largely agree with other clinical and preclinical studies of hypoxic-ischemic NE, the relative immaturity of

the animals is a limitation.3,8,30 In addition, the negative effects of endogenous SH induced by the injury do not preclude or contradict the benefits of active TH as a treatment modality,

and these two types of hypothermia should not be conflated.14 Infants with HIE who are treated with TH may present with SH but would still be expected to benefit from a prolonged period of

controlled TH.14,31 In line with this, the data presented here are unable to provide a comment on whether late-preterm infants with hypoxic-ischemic NE would or would not benefit from active

TH. This is also particularly relevant to this specific model, where LPS presensitization is required to produce a more severe injury phenotype in the ferret, but we have previously shown

that LPS presensitization negates the protective effects of TH after HI brain injury.32,33 Finally, though we hypothesize that SH might be used as an early marker of injury severity to

stratify or randomize animals to treatments, this post hoc analysis was underpowered to ascertain whether the previously described neuroprotective effects of Epo in these experiments were

modified by the severity of the injury.34 In summary, post-HI nesting temperature can act as a biomarker of injury severity in a ferret model of hypoxic-ischemic NE. In this study, cooler

post-HI temperatures were predictive of more severe outcomes in cortical pathology and development, MRI metrics, and IHC. Compared to control animals, animals displaying SH after HIH had

more severe outcomes than NT animals. These findings support post-insult temperature as a convenient method for stratifying injury severity and treatment assignment for future preclinical

studies of neonatal HI. DATA AVAILABILITY The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

REFERENCES * Organization WH. _Newborn Mortality_. https://www.who.int/news-room/fact-sheets/detail/levels-and-trends-in-child-mortality-report-2021 (2022). * Patel, S. D. et al. Therapeutic

hypothermia and hypoxia-ischemia in the term-equivalent neonatal rat: characterization of a translational preclinical model. _Pediatr. Res._ 78, 264–271, https://doi.org/10.1038/pr.2015.100

(2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Thayyil, S. et al. Hypothermia for moderate or severe neonatal encephalopathy in low-income and middle-income countries

(HELIX): a randomised controlled trial in India, Sri Lanka, and Bangladesh. _Lancet Glob. Health_ 9, e1273–e1285, https://doi.org/10.1016/S2214-109X(21)00264-3 (2021). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Jacobs, S. E. et al. Cooling for newborns with hypoxic ischaemic encephalopathy. _Cochrane Database Syst. Rev._ 1, Cd003311,

https://doi.org/10.1002/14651858.CD003311.pub3 (2013). Article  Google Scholar  * Zhou, K. Q., Davidson, J. O., Bennet, L. & Gunn, A. J. Combination treatments with therapeutic

hypothermia for hypoxic-ischemic neuroprotection. _Dev. Med. Child Neurol._ 62, 1131–1137, https://doi.org/10.1111/dmcn.14610 (2020). Article  PubMed  Google Scholar  * Victor, S. et al. New

possibilities for neuroprotection in neonatal hypoxic-ischemic encephalopathy. _Eur. J. Pediatr._ 181, 875–887, https://doi.org/10.1007/s00431-021-04320-8 (2022). Article  PubMed  Google

Scholar  * O'Mara, K. & McPherson, C. Neuroprotective agents for neonates with hypoxic-ischemic encephalopathy. _Neonatal. Netw._ 40, 406–413, https://doi.org/10.1891/11-T-755

(2021). Article  PubMed  Google Scholar  * Wu, Y. W. et al. Trial of erythropoietin for hypoxic-ischemic encephalopathy in newborns. _N. Engl. J. Med._ 387, 148–159,

https://doi.org/10.1056/NEJMoa2119660 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kao, Y. J. et al. Early neuroimaging and ultrastructural correlates of injury outcome

after neonatal hypoxic-ischaemia. _Brain Commun._ 3, fcab048, https://doi.org/10.1093/braincomms/fcab048 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Wood, T. et al.

Rectal temperature in the first five hours after hypoxia-ischemia critically affects neuropathological outcomes in neonatal rats. _Pediatr. Res._ 83, 536–544,

https://doi.org/10.1038/pr.2017.51 (2018). Article  PubMed  Google Scholar  * Busto, R. et al. Small differences in intraischemic brain temperature critically determine the extent of

ischemic neuronal injury. _J. Cereb Blood Flow Metab._ 7, 729–738, https://doi.org/10.1038/jcbfm.1987.127 (1987). Article  CAS  PubMed  Google Scholar  * Enweronu-Laryea, C. et al. Core

temperature after birth in babies with neonatal encephalopathy in a sub-Saharan African hospital setting. _J. Physiol._ 597, 4013–4024, https://doi.org/10.1113/JP277820 (2019). Article  CAS

  PubMed  Google Scholar  * Flibotte, J. et al. Blanket temperature during therapeutic hypothermia and outcomes in hypoxic ischemic encephalopathy. _J. Perinatol._ 42, 348–353,

https://doi.org/10.1038/s41372-021-01302-4 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Mietzsch, U. et al. Active cooling temperature required to achieve therapeutic

hypothermia correlates with short-term outcome in neonatal hypoxic-ischaemic encephalopathy. _J. Physiol._ 598, 415–424, https://doi.org/10.1113/JP278790 (2020). Article  CAS  PubMed  Google

Scholar  * Mietzsch U, et al. Temperature dysregulation during therapeutic hypothermia predicts long-term outcome in neonates with HIE. _J. Cereb. Blood Flow Metab_. 2023. In Press. * Wu,

T. W. et al. Cerebral lactate concentration in neonatal hypoxic-ischemic encephalopathy: in relation to time, characteristic of injury, and serum lactate concentration. _Front. Neurol._ 9,

293, https://doi.org/10.3389/fneur.2018.00293 (2018). Article  PubMed  PubMed Central  Google Scholar  * Morrison, S. F., Nakamura, K. & Madden, C. J. Central control of thermogenesis in

mammals. _Exp. Physiol._ 93, 773–797, https://doi.org/10.1113/expphysiol.2007.041848. (2008). Article  PubMed  PubMed Central  Google Scholar  * Utter, A. A. & Basso, M. A. The basal

ganglia: an overview of circuits and function. _Neurosci. Biobehav. Rev._ 32, 333–342, https://doi.org/10.1016/j.neubiorev.2006.11.003 (2008). Article  PubMed  Google Scholar  * Burnard, E.

D. & Cross, K. W. Rectal temperature in the newborn after birth asphyxia. _Br. Med. J._ 2, 1197–1199, https://doi.org/10.1136/bmj.2.5106.1197 (1958). Article  CAS  PubMed  PubMed Central

  Google Scholar  * Jayasinghe, D. Innate hypothermia after hypoxic ischaemic delivery. _Neonatology_ 107, 220–223, https://doi.org/10.1159/000369119 (2015). Article  PubMed  Google Scholar

  * Corry KA, et al. Evaluating neuroprotective effects of uridine, erythropoietin, and therapeutic hypothermia in a ferret model of inflammation-sensitized hypoxic-ischemic encephalopathy.

_Int. J. Mol. Sci_. 22 (2021). https://doi.org/10.3390/ijms22189841 * Wood T, et al. A ferret model of inflammation-sensitized late preterm hypoxic-ischemic brain injury. _J. Vis. Exp_.

(153) (2019). https://doi.org/10.3791/60131 * R Development Core Team. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical computing.

(2019). * Wieshmann, U. C. et al. Reduced anisotropy of water diffusion in structural cerebral abnormalities demonstrated with diffusion tensor imaging. _Magn. Reson. Imaging._ 17,

1269–1274, https://doi.org/10.1016/s0730-725x(99)00082-x (1999). Article  CAS  PubMed  Google Scholar  * Vijayan, V. K., Lee, Y. L. & Eng, L. F. Increase in glial fibrillary acidic

protein following neural trauma. _Mol. Chem. Neuropathol._ 13, 107–118, https://doi.org/10.1007/BF03159912 (1990). Article  CAS  PubMed  Google Scholar  * Liu, Y., Silverstein, F. S., Skoff,

R. & Barks, J. D. Hypoxic-ischemic oligodendroglial injury in neonatal rat brain. _Pediatr. Res._ 51, 25–33, https://doi.org/10.1203/00006450-200201000-00007 (2002). Article  PubMed 

Google Scholar  * Ginsberg, M. D. et al. Therapeutic modulation of brain temperature: relevance to ischemic brain injury. _Cerebrovasc Brain Metab. Rev._ 4, 189–225 (1992). CAS  PubMed 

Google Scholar  * Jang, S. H. & Kwon, H. G. Injury of the hypothalamus in patients with hypoxic-ischemic brain injury: a diffusion tensor imaging study. _Am. J. Phys. Med. Rehabil._ 97,

160–163, https://doi.org/10.1097/PHM.0000000000000813 (2018). Article  PubMed  Google Scholar  * Hutchinson, E. B. et al. Population based MRI and DTI templates of the adult ferret brain and

tools for voxelwise analysis. _Neuroimage_ 152, 575–589, https://doi.org/10.1016/j.neuroimage.2017.03.009 (2017). Article  CAS  PubMed  Google Scholar  * Gluckman, P. D. et al. Selective

head cooling with mild systemic hypothermia after neonatal encephalopathy: multicentre randomised trial. _Lancet_ 365, 663–670, https://doi.org/10.1016/S0140-6736(05)17946-X (2005). Article

  PubMed  Google Scholar  * Tsuda, K. et al. Body temperature, heart rate, and short-term outcome of cooled infants. _Ther. Hypothermia Temp. Manag._ 9, 76–85,

https://doi.org/10.1089/ther.2018.0019 (2019). Article  PubMed  PubMed Central  Google Scholar  * Falck, M. et al. Hypothermic neuronal rescue from infection-sensitised hypoxic-ischaemic

brain injury is pathogen dependent. _Dev. Neurosci._ 39, 238–247, https://doi.org/10.1159/000455838 (2017). Article  CAS  PubMed  Google Scholar  * Falck, M. et al. Neonatal systemic

inflammation induces inflammatory reactions and brain apoptosis in a pathogen-specific manner. _Neonatology_ 113, 212–220, https://doi.org/10.1159/000481980 (2018). Article  CAS  PubMed 

Google Scholar  * Osredkar, D. et al. Hypothermia does not reverse cellular responses caused by lipopolysaccharide in neonatal hypoxic-ischaemic brain injury. _Dev. Neurosci._ 37, 390–397,

https://doi.org/10.1159/000430860 (2015). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS The authors would like to thank Simar Virk and Annamarie Shearlock for

assisting in brain measurements and preparing brains for MRI. FUNDING This work was funded by the Bill and Melinda Gates Foundation. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Division of

Neonatology, Department of Pediatrics, University of Washington, Seattle, WA, USA Olivia R. White, Kylie A. Corry, Daniel H. Moralejo, Janessa B. Law, Ulrike Mietzsch, Sandra E. Juul & 

Thomas R. Wood * Department of Comparative Medicine, University of Washington, Seattle, WA, USA Jessica M. Snyder * Center on Human Development and Disability, University of Washington,

Seattle, WA, USA Sandra E. Juul & Thomas R. Wood Authors * Olivia R. White View author publications You can also search for this author inPubMed Google Scholar * Kylie A. Corry View

author publications You can also search for this author inPubMed Google Scholar * Daniel H. Moralejo View author publications You can also search for this author inPubMed Google Scholar *

Janessa B. Law View author publications You can also search for this author inPubMed Google Scholar * Jessica M. Snyder View author publications You can also search for this author inPubMed 

Google Scholar * Ulrike Mietzsch View author publications You can also search for this author inPubMed Google Scholar * Sandra E. Juul View author publications You can also search for this

author inPubMed Google Scholar * Thomas R. Wood View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Data acquisition: O.R.W., K.A.C., D.H.M.,

J.B.L., J.M.S., T.R.W. Data analysis: O.R.W., T.R.W. Interpretation: O.R.W., U.M., S.E.J., T.R.W. Manuscript drafting: O.R.W., T.R.W. Manuscript editing and revision: O.R.W., K.A.C., D.H.M.,

J.B.L., J.M.S., U.M., S.E.J.,T.R.W. Approval of final manuscript: O.R.W., K.A.C., D.H.M., J.B.L., J.M.S., U.M., S.E.J., T.R.W. CORRESPONDING AUTHOR Correspondence to Thomas R. Wood. ETHICS

DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in

published maps and institutional affiliations. RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a

publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing

agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE White, O.R., Corry, K.A., Moralejo, D.H. _et al._ Rectal temperature after hypoxia-ischemia

predicts white matter and cortical pathology in the near-term ferret. _Pediatr Res_ 95, 84–92 (2024). https://doi.org/10.1038/s41390-023-02793-x Download citation * Received: 15 March 2023 *

Revised: 11 August 2023 * Accepted: 15 August 2023 * Published: 08 September 2023 * Issue Date: January 2024 * DOI: https://doi.org/10.1038/s41390-023-02793-x SHARE THIS ARTICLE Anyone you

share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the

Springer Nature SharedIt content-sharing initiative