Treatment temperature and insult severity influence the neuroprotective effects of therapeutic hypothermia

Wood, Thomas H.; Osredkar, Damjan; Puchades, Maja; Maes, Elke; Falck, Mari; Flatebø, Torun; Walløe, Lars; Sabir, Hemmen; Thoresen, Marianne

doi:10.1038/srep23430

Cited by 91 publications

(107 citation statements)

References 76 publications

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“…In our experience HT has not been neuroprotective after mild brain injury, defined in the Vannucci model as a median area loss below 25% [51,52] . A moderate degree of injury (30-60% tissue loss) is required to see a neuroprotective effect of HT in this model [31,53] . Whether mild injury should be eligible for cooling in neonates is still debated, as the neuroprotective effect of HT has not been clarified for these patients [54] .…”

Section: Discussionmentioning

confidence: 99%

Hypothermic Neuronal Rescue from Infection-Sensitised Hypoxic-Ischaemic Brain Injury Is Pathogen Dependent

et al. 2017

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Perinatal infection increases the vulnerability of the neonatal brain to hypoxic-ischaemic (HI) injury. Hypothermia treatment (HT) does not provide neuroprotection after pre-insult inflammatory sensitisation by lipopolysaccharide (LPS), a gram-negative bacterial wall constituent. However, early-onset sepsis in term babies is caused by gram-positive species in more than 90% of cases, and neuro-inflammatory responses triggered through the gram-negative route (Toll-like receptor 4, TLR-4) are different from those induced through the gram-positive route via TLR-2. Whether gram-positive septicaemia sensitises the neonatal brain to hypoxia and inhibits the neuroprotective effect of HT is unknown. Seven-day-old Wistar rats (n = 178) were subjected to intraperitoneal injections of PAM₃CSK₄ (1 mg/kg, a synthetic TLR-2 agonist) or vehicle (0.9% NaCl). After an 8-h delay, the left carotid artery was ligated followed by 50 min of hypoxia (8% O₂) at a rectal temperature of 36°C. Pups received a 5-h treatment of normothermia (NT, 37°C) or HT (32°C) immediately after the insult. Brains were harvested after 7 days' survival for hemispheric and hippocampal area loss analyses and immunolabelling of microglia (Iba1) and hippocampal neurons (NeuN). Normothermic PAM₃CSK₄-injected animals showed significantly more brain injury than vehicle animals (p = 0.014). Compared to NT, HT significantly reduced injury in the PAM₃CSK₄-injected animals, with reduced area loss (p < 0.001), reduced microglial activation (p = 0.006), and increased neuronal rescue in the CA1 region (p < 0.001). Experimental induction of a sepsis-like condition through the gram-positive pathway sensitises the brain to HI injury. HT was highly neuroprotective after the PAM₃CSK₄-triggered injury, suggesting HT may be neuroprotective in the presence of a gram-positive infection. These results are in strong contrast to LPS studies where HT is not neuroprotective.

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Section: Discussionmentioning

confidence: 99%

Hypothermic Neuronal Rescue from Infection-Sensitised Hypoxic-Ischaemic Brain Injury Is Pathogen Dependent

et al. 2017

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“…However, although most of the studies measured environmental temperature during the insult, few monitored or reported temperature after HI. This is an important consideration since even normal, healthy neonatal rodents often show lower body temperatures in the typical laboratory nesting conditions between P7 and P14 [20] . Core temperature in neonatal rodents will be affected by the amount and composition of nesting material, position in the nest, huddling, distance from the dam, and the time since the last period of suckling.…”

Section: Discussionmentioning

confidence: 99%

In the Era of Therapeutic Hypothermia, How Well Do Studies of Perinatal Neuroprotection Control Temperature?

et al. 2016

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In the era of therapeutic hypothermia, reliable preclinical studies are integral to successfully identify neuroprotective strategies to further improve outcomes of encephalopathy at term. We reviewed preclinical neuroprotection studies reported between January 2014 and June 2016 to assess the use of effective temperature monitoring and control. As a secondary measure, we examined whether studies addressed other methodological issues such as stage of brain development, sex differences, the timing of the treatment relative to the insult, and the histological and functional endpoints used after hypoxia-ischemia. The extent and duration of temperature monitoring was highly inconsistent. Only a minority of papers monitored core (19/61; 31%) or brain temperature (3/61; 5%). Most (40/45) of the neuroprotectants either were likely to affect thermoregulation or their impact is unknown. In 85% of papers neonatal rodents were used (67% at P7); 51% of papers did not report the sex of the animals or tested the effect of potential neuroprotectants on just one sex. In 76% of studies, treatment was before or immediately after the insult (within the first 2 h), and few studies assessed long-term histological and behavioral outcomes. In conclusion, many recent preclinical neonatal studies cannot exclude the possibility that apparent neuroprotection might be related to drug-induced hypothermia or to other methodological choices. Close monitoring and control of brain temperature during, as well as for many days after, experimental hypoxia-ischemia are now critical to reliably develop new ways to improve neurodevelopmental outcomes after perinatal hypoxic-ischemic encephalopathy.

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“…We had traditionally seen a 25–50% degree of neuroprotection in moderate brain injury [5, 27]. In the mildest injuries, the method of hemispheric area loss is unable to identify small changes.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, upon adjusting the model to examine severe injury, the intrahypoxic temperature has been elevated by 1°C. Previously published data on the lack of HT neuroprotection in severe brain injury in the Vannucci model (after 60 and 66% area loss, respectively) [4, 5] comes from an experimental design where the hypoxic insult was administered at an elevated temperature of 37°C rather than the standard 36°C. In addition, a longer hypoxia time was used (150 vs. 90 min).…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, to evaluate the effect of different treatments on hippocampal neuronal loss, NeuN and DAPI-positive cells in the CA1 region of the hippocampus were counted. Aiming for a representative subset from each treatment group, the 10 animals closest to the median hemispheric area loss were selected for formal hippocampal neuron counting, as in previous publications [5]. Three non-overlapping fields, each sized 200 × 200 μm, of the CA1 region in the left hippocampi were assessed.…”

Section: Methodsmentioning

confidence: 99%

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Hypothermia Is Neuroprotective after Severe Hypoxic-Ischaemic Brain Injury in Neonatal Rats Pre-Exposed to PAM<sub>3</sub>CSK<sub>4</sub>

et al. 2018

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Background: Preclinical research on the neuroprotective effect of hypothermia (HT) after perinatal asphyxia has shown variable results, depending on comorbidities and insult severity. Exposure to inflammation increases vulnerability of the neonatal brain to hypoxic-ischaemic (HI) injury, and could be one explanation for those neonates whose injury is unexpectedly severe. Gram-negative type inflammatory exposure by lipopolysaccharide administration prior to a mild HI insult results in moderate brain injury, and hypothermic neuroprotection is negated. However, the neuroprotective effect of HT is fully maintained after gram-positive type inflammatory exposure by PAM₃CSK₄ (PAM) pre-administration in the same HI model. Whether HT is neuroprotective in severe brain injury with gram-positive inflammatory pre-exposure has not been investigated. Methods: 59 seven-day-old rat pups were subjected to a unilateral HI insult, with left carotid artery ligation followed by 90-min hypoxia (8% O₂ at T_rectal 36°C). An additional 196 pups received intraperitoneal 0.9% saline (control) or PAM_{1 mg/kg}, 8 h before undergoing the same HI insult. After randomisation to 5 h normothermia (NT₃₇_°_C) or HT₃₂_°_C, pups survived 1 week before they were sacrificed by perfusion fixation. Brains were harvested for hemispheric and hippocampal area loss analyses at postnatal day 14, as well as immunostaining for neuron count in the HIP CA1 region. Results: Normothermic PAM animals (PAM-NT) had a comparable median area loss (hemispheric: 60% [95% CI 33–66]; hippocampal: 61% [95% CI 29–67]) to vehicle animals (Veh-NT) (hemispheric: 58% [95% CI 11–64]; hippocampal: 60% [95% CI 19–68]), which is defined as severe brain injury. Furthermore, mortality was low and similar in the two groups (Veh-NT 4.5% vs. PAM-NT 6.6%). HT reduced hemispheric and hippocampal injury in the Veh group by 13 and 28%, respectively (hemispheric: p = 0.048; hippocampal: p = 0.042). HT also provided neuroprotection in the PAM group, reducing hemispheric injury by 22% (p = 0.03) and hippocampal injury by 37% (p = 0.027). Conclusion: In these experiments with severe brain injury, Toll-like receptor-2 triggering prior to HI injury does not have an additive injurious effect, and there is a small but significant neuroprotective effect of HT. HT appears to be neuroprotective over a continuum of injury severity in this model, and the effect size tapers off with increasing area loss. Our results indicate that gram-positive inflammatory exposure prior to HI injury does not negate the neuroprotective effect of HT in severe brain injury.

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Treatment temperature and insult severity influence the neuroprotective effects of therapeutic hypothermia

Cited by 91 publications

References 76 publications

Hypothermic Neuronal Rescue from Infection-Sensitised Hypoxic-Ischaemic Brain Injury Is Pathogen Dependent

Hypothermic Neuronal Rescue from Infection-Sensitised Hypoxic-Ischaemic Brain Injury Is Pathogen Dependent

In the Era of Therapeutic Hypothermia, How Well Do Studies of Perinatal Neuroprotection Control Temperature?

Hypothermia Is Neuroprotective after Severe Hypoxic-Ischaemic Brain Injury in Neonatal Rats Pre-Exposed to PAM<sub>3</sub>CSK<sub>4</sub>

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