Hypothermia is not neuroprotective after infection-sensitized neonatal hypoxic–ischemic brain injury

doi:10.1016/j.resuscitation.2013.12.006

Resuscitation

Volume 85, Issue 4, April 2014, Pages 567-572

https://doi.org/10.1016/j.resuscitation.2013.12.006 Get rights and content

Abstract

Background

Therapeutic hypothermia (HT) is the standard treatment after perinatal hypoxic–ischemic (HI) injury. Infection increases vulnerability to HI injury, but the effect of HT on lipopolysaccharide (LPS) sensitized HI brain injury is unknown.

Design/methods

P7 rat pups were injected either with vehicle or LPS, and after a 4 h delay they were exposed to left carotid ligation followed by global hypoxia inducing a unilateral stroke-like HI injury. Pups were randomized to the following treatments: (1) vehicle treated HI-pups receiving normothermia treatment (NT) (Veh-NT; n = 30); (2) LPS treated HI-pups receiving NT treatment (LPS-NT; n = 35); (3) vehicle treated HI-pups receiving HT treatment (Veh-HT; n = 29); or (4) LPS treated HI-pups receiving HT treatment (LPS-HT; n = 46). Relative area loss of the left/right hemisphere and the areas of hippocampi were measured at P14.

Results

Mean brain area loss in the Veh-NT group was 11.2 ± 14%. The brain area loss in LPS-NT pups was 29.8 ± 17%, which was significantly higher than in the Veh-NT group (p = 0.002). The Veh-HT group had a significantly smaller brain area loss (5.4 ± 6%), when compared to Veh-NT group (p = 0.043). The LPS-HT group showed a brain area loss of 32.5 ± 16%, which was significantly higher than in the Veh-HT group (p < 0.001). LPS-HT group also had significantly smaller size of the left hippocampus, which was not found in other groups. LPS-sensitization significantly decreased the sizes of the right, unligated-hemispheres, independent of post-HI treatment.

Conclusions

Therapeutic hypothermia is not neuroprotective in this LPS-sensitized unilateral stroke-like HI brain injury model in newborn rats. Lack of neuroprotection was particularly seen in the hippocampus. Pre-insult exposure to LPS also induced brain area loss in the unligated hemisphere, which is normally not affected in this model.

Introduction

Perinatal hypoxic–ischemic (HI) injury is one of the major causes of long-term neurological disability or death in term newborns. Infection is known to be a major confounding factor for neonatal morbidity and mortality, especially in developing countries.¹ Perinatal infection increases the vulnerability of the newborn brain to HI.2, 3, 4, 5 In an established neonatal rat model of unilateral HI brain injury,⁶ a longer period of exposure to hypoxia results in increased brain injury.⁷ In the same model, increased injury can also follow a shorter period of hypoxia, if animals have been subjected to a mild infectious stimulus such as pre-exposure to bacterial lipopolysaccharide (LPS) before HI.³

Therapeutic hypothermia (HT) is the standard treatment for term infants after perinatal HI injury,⁸ as it has been shown to significantly reduce mortality and neurodevelopmental disability in survivors.⁹ However, around 50% of cooled asphyxiated newborns still suffer poor outcomes,⁹ some of which may have been exposed to perinatal infection.¹⁰ It has been shown that HT influences many cascades that follow a HI injury, including inflammatory and anti-inflammatory pathways,11, 12, 13 but this influence might be different following pre-exposure to an infectious stimulus.

This study was designed to investigate the neuroprotective effect of HT in a double-hit neonatal model of LPS-sensitized HI brain injury, which has not been previously investigated.

Section snippets

Procedures

All experiments were approved by the University of Oslo's animal ethics research committee. Experiments were performed on 7 day old (P7) Wistar rats (Charles River, Sulzfeld, Germany) of both sexes, randomized across litter, sex and weight. All pups were kept in an animal facility with a 12:12 h dark/light cycle at 19–21 °C environmental temperature with food and water ad libitum, and were weighed and checked for health daily.

At the start of every experiment, we injected the animals according to

Results

Of 140 rat pups, 6 pups died during ligation, 3 between ligation and the hypoxic insult, 21 during the 50 min of hypoxia, 1 during the 5 h treatment period and 9 between P8 and P14. Of the 9 that died after the treatment period, 7 received LPS (2 in the NT LPS group and 5 in the HT LPS group). The twelve sentinel pups carrying temperature probes were excluded from further analysis, because the stress of carrying the probe could influence the outcome.¹⁸ The final number of animals included in

Discussion

We have shown that sensitizing a neonatal unilateral HI brain injury model with LPS injection prior to the HI injury increases the injury about 3-fold at NT. The new finding of the current study is that HT is not neuroprotective after LPS-sensitized HI brain injury as compared to HT without LPS.

Therapeutic hypothermia is one of the most intensively studied neuroprotective strategies to date. It is recommended by the ILCOR guidelines for two conditions, adult out of hospital cardiac arrest¹⁹ and

Conflict of interest statement

I declare that neither me or any of the co-authors are in conflict of interest.

Disclosures

None.

Acknowledgements

This study was supported by the Norwegian Research Council (NFR 214356/F20). We thank Professor Lars Walløe for advice on statistical analysis and Dr Jonathan Davis for valuable comments on the manuscript.

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Hypoxic-ischemic (HI) brain injury is a major contributor to neurodevelopmental morbidities. Inter-alpha inhibitor proteins (IAIPs) have neuroprotective effects on HI-related brain injury in neonatal rats. However, the effects of treatment with IAIPs on sequential behavioral, MRI, and histopathological abnormalities in the young adult brain after treatment with IAIPs in neonates remain to be determined. The objective of this study was to examine the neuroprotective effects of IAIPs at different neurodevelopmental stages from newborn to young adults after exposure of neonates to HI injury. IAIPs were given as 11-sequential 30-mg/kg doses to postnatal (P) day 7–21 rats after right common carotid artery ligation and exposure to 90 min of 8% oxygen. The resulting brain edema and injury were examined by T₂-weighted magnetic resonance imaging (MRI) and cresyl violet staining, respectively. The mean T₂ values of the ipsilateral hemisphere from MRI slices 6 to 10 were reduced in IAIP-treated HI males + females on P8, P9, and P10 and females on P8, P9, P10, and P14. IAIP treatment reduced hemispheric volume atrophy by 44.5 ± 29.7% in adult male + female P42 rats and improved general locomotor abilities measured by the righting reflex over time at P7.5, P8, and P9 in males + females and males and muscle strength/endurance measured by wire hang on P16 in males + females and females. IAIPs provided beneficial effects during the learning phase of the Morris water maze with females exhibiting beneficial effects. IAIPs confer neuroprotection from HI-related brain injury in neonates and even in adult rats and beneficial MRI and behavioral benefits in a sex-dependent manner.
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In term and near-term neonates with neonatal encephalopathy, therapeutic hypothermia protocols are well established. The current focus is on how to improve outcomes further and the challenge is to find safe and complementary therapies that confer additional protection, regeneration or repair in addition to cooling. Following hypoxia-ischemia, brain injury evolves over three main phases (latent, secondary and tertiary), each with a different brain energy, perfusion, neurochemical and inflammatory milieu. While therapeutic hypothermia has targeted the latent and secondary phase, we now need therapies that cover the continuum of brain injury that spans hours, days, weeks and months after the initial event. Most agents have several therapeutic actions but can be broadly classified under a predominant action (e.g., free radical scavenging, anti-apoptotic, anti-inflammatory, neuroregeneration, and vascular effects). Promising early/secondary phase therapies include Allopurinol, Azithromycin, Exendin-4, Magnesium, Melatonin, Noble gases and Sildenafil. Tertiary phase agents include Erythropoietin, Stem cells and others. We review a selection of promising therapeutic agents on the translational pipeline and suggest a framework for neuroprotection and neurorestoration that targets the evolving injury.
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Citation Excerpt :
Perinatal infection may lead to chorioamnionitis or funisitis, which not automatically lead to brain injury. As shown in newborn rodents, an inflammatory stimulus before hypoxia-ischemia can exacerbate HI brain injury [79,80]. However, the time interval between the inflammatory stimulus and the onset of HI is crucial.
Therapeutic hypothermia (TH) is now well established to improve intact survival after neonatal encephalopathy (NE). However, many questions could not be addressed by the randomized controlled trials. Should late preterm newborns with NE be cooled? Is cooling beneficial for mild NE? Is the current therapeutic time window optimal, or could it be shortened or prolonged? Will either milder or deeper hypothermia be effective? Does infection/inflammation exposure in the perinatal period in combination with NE offer potentially beneficial preconditioning or might it obviate hypothermic neuroprotection? In the present review, we dissect the evidence, for whom, when and how can TH best be delivered, and highlight areas that need further research.
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
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Although therapeutic hypothermia reduces death or disability after neonatal encephalopathy in high-income countries, its safety and efficacy in low-income and middle-income countries is unclear. We aimed to examine whether therapeutic hypothermia alongside optimal supportive intensive care reduces death or moderate or severe disability after neonatal encephalopathy in south Asia.
We did a multicountry open-label, randomised controlled trial in seven tertiary neonatal intensive care units in India, Sri Lanka, and Bangladesh. We enrolled infants born at or after 36 weeks of gestation with moderate or severe neonatal encephalopathy and a need for continued resuscitation at 5 min of age or an Apgar score of less than 6 at 5 min of age (for babies born in a hospital), or both, or an absence of crying by 5 min of age (for babies born at home). Using a web-based randomisation system, we allocated infants into a group receiving whole body hypothermia (33·5°C) for 72 h using a servo-controlled cooling device, or to usual care (control group), within 6 h of birth. All recruiting sites had facilities for invasive ventilation, cardiovascular support, and access to 3 Tesla MRI scanners and spectroscopy. Masking of the intervention was not possible, but those involved in the magnetic resonance biomarker analysis and neurodevelopmental outcome assessments were masked to the allocation. The primary outcome was a combined endpoint of death or moderate or severe disability at 18–22 months, assessed by the Bayley Scales of Infant and Toddler Development (third edition) and a detailed neurological examination. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, NCT02387385.
We screened 2296 infants between Aug 15, 2015, and Feb 15, 2019, of whom 576 infants were eligible for inclusion. After exclusions, we recruited 408 eligible infants and we assigned 202 to the hypothermia group and 206 to the control group. Primary outcome data were available for 195 (97%) of the 202 infants in the hypothermia group and 199 (97%) of the 206 control group infants. 98 (50%) infants in the hypothermia group and 94 (47%) infants in the control group died or had a moderate or severe disability (risk ratio 1·06; 95% CI 0·87–1·30; p=0·55). 84 infants (42%) in the hypothermia group and 63 (31%; p=0·022) infants in the control group died, of whom 72 (36%) and 49 (24%; p=0·0087) died during neonatal hospitalisation. Five serious adverse events were reported: three in the hypothermia group (one hospital readmission relating to pneumonia, one septic arthritis, and one suspected venous thrombosis), and two in the control group (one related to desaturations during MRI and other because of endotracheal tube displacement during transport for MRI). No adverse events were considered causally related to the study intervention.
Therapeutic hypothermia did not reduce the combined outcome of death or disability at 18 months after neonatal encephalopathy in low-income and middle-income countries, but significantly increased death alone. Therapeutic hypothermia should not be offered as treatment for neonatal encephalopathy in low-income and middle-income countries, even when tertiary neonatal intensive care facilities are available.
National Institute for Health Research, Garfield Weston Foundation, and Bill & Melinda Gates Foundation.
For the Hindi, Malayalam, Telugu, Kannada, Singhalese, Tamil, Marathi and Bangla translations of the abstract see Supplementary Materials section.
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^☆: A Spanish translated version of the abstract of this article appears as Appendix in the final online version at http://dx.doi.org/10.1016/j.resuscitation.2013.12.006.

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Experimental paperHypothermia is not neuroprotective after infection-sensitized neonatal hypoxic–ischemic brain injury☆

Abstract

Background

Design/methods

Results

Conclusions

Introduction

Section snippets

Procedures

Results

Discussion

Conflict of interest statement

Disclosures

Acknowledgements

Semin Perinatol

Resuscitation

Lancet Neurol

Lancet Neurol

Semin Fetal Neonatal Med

J Neuroimmunol

Int Health

Lipopolysaccharide sensitizes neonatal hypoxic–ischemic brain injury in a MyD88-dependent manner

J Immunol

Bacterial endotoxin sensitizes the immature brain to hypoxic–ischaemic injury

Eur J Neurosci

Systemic and neuropathologic effects of E. coli endotoxin in neonatal dogs

Pediatr Res

White matter injury following systemic endotoxemia or asphyxia in the fetal sheep

Neurochem Res

The influence of immaturity on hypoxic–ischemic brain damage in the rat

Ann Neurol

Immediate hypothermia is not neuroprotective after severe hypoxia–ischemia and is deleterious when delayed by 12 hours in neonatal rats

Stroke

Part 11: neonatal resuscitation: 2010 international consensus on cardiopulmonary resuscitation and emergency cardiovascular care science with treatment recommendations

Circulation

Cooling for newborns with hypoxic ischaemic encephalopathy

Cochrane Database Syst Rev

Therapeutic hypothermia in the prevention of hypoxic–ischaemic encephalopathy: new categories to be enrolled

J Matern Fetal Neonatal Med

Experimental paper
Hypothermia is not neuroprotective after infection-sensitized neonatal hypoxic–ischemic brain injury☆