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Hippocampal immunohistochemistry.: Representative CA1 hippocampal regions of interest from each treatment group (indicated in the top left hand side of each image). Arrows show typical viable pyramidal neurons with large, round nuclei (DAPI, blue), and NeuN co-staining (red). Scale bar (bottom right) represents 50 μm.
Source publication
Therapeutic hypothermia (HT) is standard care for moderate and severe neonatal hypoxic-ischaemic encephalopathy (HIE), the leading cause of permanent brain injury in term newborns. However, the optimal temperature for HT is still unknown, and few preclinical studies have compared multiple HT treatment temperatures. Additionally, HT may not benefit...
Contexts in source publication
Context 1
... CA1 pyramidal neurons was seen in the HT33.5, HT32, HT30, and HT26 groups (Fig. 2B). Within the therapeutic range of HT, there was a trend towards greater neuroprotection of pyramidal neurons in the HT33.5 and HT32 groups compared to the HT30 group (Table 2). Representative regions of interest (ROIs) from each treatment group are shown in Fig. 3. Total CA1 neuron count on the right (contralateral) side of the brain ...
Context 2
... N = Male Weight P7 (g) Weight P14 (g) Weight Gain (g) Weight gain (%) Anaesthesia time similar across all six groups, with no effect of treatment temperature or ipsilateral injury severity (Supplementary Figure S2). Representative hippocampal ROIs from control P14 animals are shown in Supplementary Figure S3. ...
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Background
Several low-cost methods are used in resource-limited settings to provide therapeutic hypothermia in asphyxiated neonates. There is inadequate data about their efficacy and safety.
Methods
This is a retrospective study comparing two low-cost cooling methods—frozen gel packs (FGP) and phase changing material (PCM).
Results
There were 23...
Objective Hypothermia is considered the treatment of choice for newborns with hypoxic-ischemic encephalopathy. Even though currently no consensus exists regarding the opportunity to extend this treatment also to newborns with sudden unexpected postnatal collapse (SUPC), this treatment is frequently adopted. The aim of the study was to evaluate whet...
Background
Cerebral lactate concentration can remain detectable in neonatal hypoxic-ischemic encephalopathy (HIE) after hemodynamic stability. The temporal resolution of regional cerebral lactate concentration in relation to the severity or area of injury is unclear. Furthermore, the interplay between serum and cerebral lactate in neonatal HIE has...
Objective:
To quantify the prognostic value of neonatal brain magnetic resonance imaging (MRI) in neonatal hypoxic-ischemic encephalopathy.
Methods:
Meta-analysis of studies with ≥35-week neonates with hypoxic-ischemic encephalopathy who underwent brain MRI within age 4 weeks and had neurodevelopmental follow-up for at least 12 months.
Results:...
Citations
... The 3-h treatment with hypothermia was based upon previous studies in neonatal rats [49,50]. Although we have demonstrated that hIAIPs exert neuroprotective effects even after exposure to severe HI [27], moderate exposure (90 min of 8% oxygen and balanced nitrogen) was utilized in the current study because previous work has shown that hypothermia may not be protective after exposure to severe HI in neonatal rats [83,84]. ...
Therapeutic hypothermia is the standard of care for hypoxic-ischemic (HI) encephalopathy. Inter-alpha Inhibitor Proteins (IAIPs) attenuate brain injury after HI in neonatal rats. Human (h) IAIPs (60 mg/kg) or placebo (PL) were given 15 min, 24 and 48 h to postnatal (P) day-7 rats after carotid ligation and 8% oxygen for 90 min with (30 °C) and without (36 °C) exposure to hypothermia 1.5 h after HI for 3 h. Hemispheric volume atrophy (P14) and neurobehavioral tests including righting reflex (P8–P10), small open field (P13–P14), and negative geotaxis (P14) were determined. Hemispheric volume atrophy in males was reduced (P < 0.05) by 41.9% in the normothermic-IAIP and 28.1% in the hypothermic-IAIP compared with the normothermic-PL group, and in females reduced (P < 0.05) by 30.3% in the normothermic-IAIP, 45.7% in hypothermic-PL, and 55.2% in hypothermic-IAIP compared with the normothermic-PL group after HI. Hypothermia improved (P < 0.05) the neuroprotective effects of hIAIPs in females. The neuroprotective efficacy of hIAIPs was comparable to hypothermia in female rats (P = 0.183). Treatment with hIAIPs, hypothermia, and hIAIPs with hypothermia decreased (P < 0.05) the latency to enter the peripheral zone in the small open field test in males. We conclude that hIAIPs provide neuroprotection from HI brain injury that is comparable to the protection by hypothermia, hypothermia increases the effects of hIAIPs in females, and hIAIPs and hypothermia exhibit some sex-related differential effects.
... Hence, a small difference in core temperature may tip the balance of benefit (neuroprotection) versus harm (systemic complications) when performing TH as a neuroprotective measure for HIE. (4)(5)(6) Preclinical models of neuronal rescue have assessed cerebral hypothermia by invasive measurements of dural or intracerebral temperature(7-10); however, brain temperature monitoring during TH is not performed in the clinical setting. Non-invasive methods of brain temperature monitoring are being developed but remain confined to research use. ...
... Modulation of cerebral temperature to different hypothermic depths impacts neuronal rescue and neurodevelopmental outcome in animal models (4,(37)(38)(39) and clinical trials (3). From preclinical studies, the general relationship between neuronal loss and depth of hypothermia is proposed to be a U-shaped curve.(4) ...
... Clinically, results seem more consistent, and male sex is considered a risk factor [23]. Further, the neuroprotective effect of therapies may differ depending on sex [24], just as may the neurogenic response of some treatments [25,26]. ...
The need for new and effective treatments for neonates suffering from hypoxia–ischemia is urgent, as the only implemented therapy in clinics is therapeutic hypothermia, only effective in 50% of cases. Cannabinoids may modulate neuronal development and brain plasticity, but further investigation is needed to better describe their implication as a neurorestorative therapy after neonatal HI. The cannabinoid URB447, a CB1 antagonist/CB2 agonist, has previously been shown to reduce brain injury after HI, but it is not clear whether sex may affect its neuroprotective and/or neurorestorative effect. Here, URB447 strongly reduced brain infarct, improved neuropathological score, and augmented proliferative capacity and neurogenic response in the damaged hemisphere. When analyzing these effects by sex, URB447 ameliorated brain damage in both males and females, and enhanced cell proliferation and the number of neuroblasts only in females, thus suggesting a neuroprotective effect in males and a double neuroprotective/neurorestorative effect in females.
... After a 90 min recovery period (in which the animals were returned to their mothers), the rats were submitted to a hypoxic atmosphere (8% O2 balanced in 92% N2) at 37 °C for 60 min. In general, at this developmental stage, the parameters used in this study caused a moderate degree of injury [80] and they were chosen since hypothermia may not be effective in cases of severe HI [81,82]. The sham-operated rats had neither a common carotid artery ligation nor hypoxia exposure. ...
... After a 90 min recovery period (in which the animals were returned to their mothers), the rats were submitted to a hypoxic atmosphere (8% O 2 balanced in 92% N 2 ) at 37 • C for 60 min. In general, at this developmental stage, the parameters used in this study caused a moderate degree of injury [80] and they were chosen since hypothermia may not be effective in cases of severe HI [81,82]. The sham-operated rats had neither a common carotid artery ligation nor hypoxia exposure. ...
Neonatal hypoxic-ischemic (HI) encephalopathy (HIE) in term newborns is a leading cause of mortality and chronic disability. Hypothermia (HT) is the only clinically available therapeutic intervention; however, its neuroprotective effects are limited. Lactoferrin (LF) is the major whey protein in milk presenting iron-binding, anti-inflammatory and anti-apoptotic properties and has been shown to protect very immature brains against HI damage. We hypothesized that combining early oral administration of LF with whole body hypothermia could enhance neuroprotection in a HIE rat model. Pregnant Wistar rats were fed an LF-supplemented diet (1 mg/kg) or a control diet from (P6). At P7, the male and female pups had the right common carotid artery occluded followed by hypoxia (8% O2 for 60′) (HI). Immediately after hypoxia, hypothermia (target temperature of 32.5–33.5 °C) was performed (5 h duration) using Criticool®. The animals were divided according to diet, injury and thermal condition. At P8 (24 h after HI), the brain neurochemical profile was assessed using magnetic resonance spectroscopy (1H-MRS) and a hyperintense T2W signal was used to measure the brain lesions. The mRNA levels of the genes related to glutamatergic excitotoxicity, energy metabolism and inflammation were assessed in the right hippocampus. The cell markers and apoptosis expression were assessed using immunofluorescence in the right hippocampus. HI decreased the energy metabolites and increased lactate. The neuronal–astrocytic coupling impairments observed in the HI groups were reversed mainly by HT. LF had an important effect on astrocyte function, decreasing the levels of the genes related to glutamatergic excitotoxicity and restoring the mRNA levels of the genes related to metabolic support. When combined, LF and HT presented a synergistic effect and prevented lactate accumulation, decreased inflammation and reduced brain damage, pointing out the benefits of combining these therapies. Overall, we showed that through distinct mechanisms lactoferrin can enhance neuroprotection induced by HT following neonatal brain hypoxia-ischemia.
... Across multiple species, there are windows of temperature reduction that are beneficial (112). A study with neonatal rodents found that temperature up to a 3.5 ℃ reduction yielded neuroprotection after moderate injury (113). Similarly, a study with neonatal pigs found a therapeutic window between a 3.5 to a 5 ℃ reduction, whereas a 8.5 ℃ reduction was detrimental (114). ...
Background and objective:
Hypoxic-ischemic encephalopathy (HIE) is a leading cause of death and disability worldwide. Therapeutic hypothermia (TH) represents a significant achievement in the translation of scientific research to clinical application, but it is currently the only neuroprotective treatment for HIE. This review aims to revisit the use of TH for HIE and its longitudinal impact on patient outcomes to readers new to the field of HIE. We discuss how emerging therapies address the broader pathophysiology of injury progression in the neonatal brain days to years after HIE.
Methods:
We included full articles and book chapters published in English on PubMed with references to "hypoxic ischemic encephalopathy", "birth asphyxia", "therapeutic hypothermia", or "neonatal encephalopathy". We limited our review to outcomes on term infants and to new therapeutics that are in the second phase of clinical trials.
Key content and findings:
Despite the use of TH for HIE, mortality remains high. Analysis of longitudinal studies reveals a high incidence of ongoing disability even with the implementation of TH. New therapeutics addressing the secondary phase and the less understood tertiary phase of brain injury are in clinical trials as adjunctive treatments to TH to support additional neurological repair and regeneration.
Conclusions:
TH successfully improves outcomes after HIE, and it continues to be optimized. Larger studies are needed to understand its use in mild cases of HIE and if certain factors, such as sex, affect long term outcomes. TH primarily acts in the initial phases of injury, while new pharmaceutical therapies target additional injury pathways into the tertiary phases of injury. This may allow for more effective approaches to treatment and improvement of long-term functional outcomes after HIE.
... All animal experiment protocols were approved by the animal protection committee from the State Environment Agency of North Rhine-Westphalia, Germany (LANUV) (Approval No. AZ 81-02.04.2018.A166), and all experiments were performed following relevant guidelines and regulations and in compliance with the ARRIVE guidelines. We used our established rat model of unilateral hypoxic-ischemic brain injury, in which we have previously shown significant neuroprotection using HT and additional treatments with immediate and delayed treatment onset following HI [17][18][19][20][21] . ...
... As previously described, all animals for each experiment were randomized across litter, sex, and weight before the experiments commenced and all read-out analysis were performed by observers blinded to the different treatments. All animals underwent unilateral ligation of the left common carotid artery under general anaesthesia as previously described in detail in our previous publications [17][18][19][20][21] . Animals were kept at physiological temperature whilst handling and ligation, as previously described [17][18][19][20][21] . ...
... All animals underwent unilateral ligation of the left common carotid artery under general anaesthesia as previously described in detail in our previous publications [17][18][19][20][21] . Animals were kept at physiological temperature whilst handling and ligation, as previously described [17][18][19][20][21] . After ligation of all pups, pups were exposed to 8% oxygen (hypoxia) for 90 min at a rectal temperature (T rectal ) of 36.0 °C in a temperature-controlled chamber, resulting in a unilateral moderate hypoxic-ischemic insult. ...
Intrapartum hypoxia–ischemia leading to neonatal encephalopathy (NE) results in significant neonatal mortality and morbidity worldwide, with > 85% of cases occurring in low- and middle-income countries (LMIC). Therapeutic hypothermia (HT) is currently the only available safe and effective treatment of HIE in high-income countries (HIC); however, it has shown limited safety or efficacy in LMIC. Therefore, other therapies are urgently required. We aimed to compare the treatment effects of putative neuroprotective drug candidates following neonatal hypoxic-ischemic (HI) brain injury in an established P7 rat Vannucci model. We conducted the first multi-drug randomized controlled preclinical screening trial, investigating 25 potential therapeutic agents using a standardized experimental setting in which P7 rat pups were exposed to unilateral HI brain injury. The brains were analysed for unilateral hemispheric brain area loss after 7 days survival. Twenty animal experiments were performed. Eight of the 25 therapeutic agents significantly reduced brain area loss with the strongest treatment effect for Caffeine, Sonic Hedgehog Agonist (SAG) and Allopurinol, followed by Melatonin, Clemastine, ß-Hydroxybutyrate, Omegaven, and Iodide. The probability of efficacy was superior to that of HT for Caffeine, SAG, Allopurinol, Melatonin, Clemastine, ß-hydroxybutyrate, and Omegaven. We provide the results of the first systematic preclinical screening of potential neuroprotective treatments and present alternative single therapies that may be promising treatment options for HT in LMIC.
... 7,8,16 In contrast, histological analysis revealed two subgroups within the KO group, one subgroup with severe injury and associated marked microglia activation (58% KO) and another with injury limited to the caudate and lack/minor signs of microglial activation (42% KO). We did not lump the two KO subgroups together and focused only on severely injured KO subgroup as a translationally relevant approach, as studies in human neonates with hypoxic-ischemic encephalopathy (HIE) and arterial stroke demonstrate that the severity of initial injury critically affects responsiveness to therapeutic approaches, 23 justifying the need to study infants with severe and moderate injury separately. We demonstrate long-lasting improved functional outcome in KO pups with severe injury. ...
Arterial ischemic stroke is common in neonates—1 per 2,300–5,000 births—and therapeutic targets remain insufficiently defined. Sphingosine-1-phosphate receptor 2 (S1PR2), a major regulator of the CNS and immune systems, is injurious in adult stroke. Here, we assessed whether S1PR2 contributes to stroke induced by 3 h transient middle cerebral artery occlusion (tMCAO) in S1PR2 heterozygous (HET), knockout (KO), and wild type (WT) postnatal day 9 pups. HET and WT of both sexes displayed functional deficits in Open Field test whereas injured KO at 24 h reperfusion performed similarly to naives. S1PR2 deficiency protected neurons, attenuated infiltration of inflammatory monocytes, and altered vessel-microglia interactions without reducing increased cytokine levels in injured regions at 72 h. Pharmacologic inhibition of S1PR2 after tMCAO by JTE-013 attenuated injury 72 h after tMCAO. Importantly, the lack of S1PR2 alleviated anxiety and brain atrophy during chronic injury. Altogether, we identify S1PR2 as a potential new target for mitigating neonatal stroke.
... As demonstrated previously, TH was neuroprotective after HI in our model and reduced the area of injury (Figure 2a-c) [10,52,53]. However, the immune response of peripheral cells in the injured brain, particularly after TH, remains unknown. ...
... During all the experiments, the temperature of the rat pups was controlled by the sentinel pup via a rectal probe (IT-21, Physitemp Instruments, Clifton, NJ, USA), connected to a servocontrolled cooling machine and a cooling mat (CritiCool, MTRE, Yavne, Israel). The sentinel pup maintained the nesting temperature of the P7 rat pups [53] or treatment temperatures during the experiments (see below). The standardized HI Vannucci rat model was used, as previously described [46,94,97]. ...
... The optical density and hemispheric area were analyzed using the ImageJ software. The ipsilateral side was compared to the contralateral side, and the area loss of the ligated side was calculated using the formula (1 − (area left/area right) × 100), as previously described [53,97,98]. were performed by observers blinded to the different treatments [52,98]. ...
The peripheral immune system plays a critical role in neuroinflammation of the central nervous system after an insult. Hypoxic-ischemic encephalopathy (HIE) induces a strong neuroinflammatory response in neonates, which is often associated with exacerbated outcomes. In adult models of ischemic stroke, neutrophils infiltrate injured brain tissue immediately after an ischemic insult and aggravate inflammation via various mechanisms, including neutrophil extracellular trap (NETs) formation. In this study, we used a neonatal model of experimental hypoxic-ischemic (HI) brain injury and demonstrated that circulating neutrophils were rapidly activated in neonatal blood. We observed an increased infiltration of neutrophils in the brain after exposure to HI. After treatment with either normothermia (NT) or therapeutic hypothermia (TH), we observed a significantly enhanced expression level of the NETosis marker Citrullinated H3 (Cit-H3), which was significantly more pronounced in animals treated with TH than in those treated with NT. NETs and NLR family pyrin domain containing 3 (NLRP-3) inflammasome assembly are closely linked in adult models of ischemic brain injury. In this study, we observed an increase in the activation of the NLRP-3 inflammasome at the time points analyzed, particularly immediately after TH, when we observed a significant increase in NETs structures in the brain. Together, these results suggest the important pathological functions of early arriving neutrophils and NETosis following neonatal HI, particularly after TH treatment, which is a promising starting point for the development of potential new therapeutic targets for neonatal HIE.
... Therefore, we combined the three hIAIP-treated groups into one hIAIP treatment group ( Figure 8) for the infarct volume analysis and confirmed that treatment with hIAIPs reduced the infarct volumes in the hemisphere, cortex, and striatum of the male neonatal rats, but not the female neonatal rats, after exposure to severe HI. In this context, it is important to emphasize that hypothermia was not neuroprotective in previous studies when applied to neonatal rats after exposure to severe HI [39,41,42]. The severity of the HI insults and the 37 °C temperature used during the induction of HI were similar in the present study and the former work [39]. ...
... In the present study, the NNT to reduce infarct volumes in the brain to below 30% of the hemisphere was eight for females and four for males. Hypothermia treatment has been reported to have an NNT of 10 to achieve equivalent infarct parameters in a similar preclinical rodent model of injury [42]. Treatment with therapeutic hypothermia after exposure of full-term infants to HIE has been reported to have an NNT of 7-14 over a range of mortalities and neurodevelopmental parameters [6,45]. ...
Hypoxia–ischemia (HI)-related brain injury is an important cause of morbidity and long-standing disability in newborns. We have previously shown that human plasma-derived inter-alpha inhibitor proteins (hIAIPs) attenuate HI-related brain injury in neonatal rats. The optimal dose of hIAIPs for their neuroprotective effects and improvement in behavioral outcomes remains to be determined. We examined the efficacy of 30, 60, or 90 mg/kg of hIAIPs administered to neonatal rats after exposure to HI for 2 h. Postnatal day 7 (P7) Wistar rats were exposed to either sham-surgery or unilateral HI (right carotid artery ligation, 2 h of 8% O2) brain injury. A placebo, 30, 60, or 90 mg/kg of hIAIPs were injected intraperitoneally at 0, 24 and 48 h after HI (n = 9–10/sex). We carried out the following behavioral analyses: P8 (righting reflex), P9 (negative geotaxis) and P10 (open-field task). Rats were humanely killed on P10 and their brains were stained with cresyl violet. Male extension/contraction responses and female righting reflex times were higher in the HI placebo groups than the sham groups. Female open-field exploration was lower in the HI placebo group than the sham group. hIAIPs attenuated these behavioral deficits. However, the magnitude of the responses did not vary by hIAIP dose. hIAIPs reduced male brain infarct volumes in a manner that correlated with improved behavioral outcomes. Increasing the hIAIP dose from 30 to 90 mg/kg did not further accentuate the hIAIP-related decreases in infarct volumes. We conclude that larger doses of hIAIPs did not provide additional benefits over the 30 mg/kg dose for behavior tasks or reductions in infarct volumes in neonatal rats after exposure to severe HI.
... Deficits on spatial and working memory tasks have also been validated in P7 HI rats (Morris Water Maze task, Non-Spatial Maze, delayed-match-to-place maze tasks) [93,96,97,[113][114][115][116][117], as well as deficits in visual attention [114,118,119]. Perhaps based on this extensive literature, most pre-clinical neuroprotective HI studies use a P7 HI rat model (Rice-Vannucci) [88,104,105,[120][121][122][123][124][125][126][127][128][129][130][131][132]. ...
... Matched littermates received HI followed by normothermic conditions, or sham treatment with comparable hypothermia or normothermia. Results showed that this cooling intervention was not only ineffective, but it was also deleterious to both sham and P6 HI rats as measured by behavioral and neuroanatomical outcomes [129,149]. Specifically, cooled sham males had worse scores on a Silent Gap acoustic task compared to normothermic sham males, and significantly worse scores on Non-Spatial Water Maze (p < 0.01) [149]. These findings have important implications for therapeutic intervention in at-risk preterm human populations, and certainly promote caution in the application of existing hypothermia protocols to at-risk preterm infants. ...
Infants born prematurely have an increased risk of experiencing brain injury, specifically injury caused by Hypoxia Ischemia (HI). There is no approved treatment for preterm infants, in contrast to term infants that experience Hypoxic Ischemic Encephalopathy (HIE) and can be treated with hypothermia. Given this increased risk and lack of approved treatment, it is imperative to explore and model potential treatments in animal models of preterm injury. Hypothermia is one potential treatment, though cooling to current clinical standards has been found to be detrimental for preterm infants. However, mild hypothermia may prove useful. Caffeine is another treatment that is already used in preterm infants to treat apnea of prematurity, and has shown neuroprotective effects. Both of these treatments show sex differences in behavioral outcomes and neuroprotective effects, which are critical to explore when working to translate from animal to human. The effects and research history of hypothermia, caffeine and how sex affects these treatment outcomes will be explored further in this review article.
