Neutrophil Extracellular Traps Release following Hypoxic-Ischemic Brain Injury in Newborn Rats Treated with Therapeutic Hypothermia.

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.

Hypothermia Is Neuroprotective after Severe Hypoxic-Ischaemic Brain Injury in Neonatal Rats Pre-Exposed to PAM3CSK4.

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 Trectal 36°C). An additional 196 pups received intraperitoneal 0.9% saline (control) or PAM1 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.

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

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.

Hypothermia Does Not Reverse Cellular Responses Caused by Lipopolysaccharide in Neonatal Hypoxic-Ischaemic Brain Injury.

INTRODUCTION: Bacterial lipopolysaccharide (LPS) injection prior to hypoxia-ischaemia significantly increases hypoxia-ischaemic brain injury in 7-day-old (P7) rats. In addition, therapeutic hypothermia (HT) is not neuroprotective in this setting. However, the mechanistic aspects of this therapeutic failure have yet to be elucidated. This study was designed to investigate the underlying cellular mechanisms in this double-hit model of infection-sensitised hypoxia-ischaemic brain injury. MATERIAL AND METHODS: P7 rat pups were injected with either vehicle or LPS, and after a 4-hour delay were exposed to left carotid ligation followed by global hypoxia inducing a unilateral stroke-like hypoxia-ischaemic injury. Pups were randomised to the following treatments: (1) vehicle-treated pups receiving normothermia treatment (NT) (Veh-NT; n = 40), (2) LPS-treated pups receiving NT treatment (LPS-NT; n = 40), (3) vehicle-treated pups receiving HT treatment (Veh-HT; n = 38) and (4) LPS-treated pups receiving HT treatment (LPS-HT; n = 35). On postnatal day 8 or 14, Western blot analysis or immunohistochemistry was performed to examine neuronal death, apoptosis, astrogliosis and microglial activation. RESULTS: LPS sensitisation prior to hypoxia-ischaemia significantly exacerbated apoptotic neuronal loss. NeuN, a neuronal biomarker, was significantly reduced in the LPS-NT and LPS-HT groups (p = 0.008). Caspase-3 activation was significantly increased in the LPS-sensitised groups (p < 0.001). Additionally, a significant increase in astrogliosis (glial fibrillary acidic expression, p < 0.001) was seen, as well as a trend towards increased microglial activation (Iba 1 expression, p = 0.051) in LPS-sensitised animals. Treatment with HT did not counteract these changes. CONCLUSION: LPS-sensitised hypoxia-ischaemic brain injury in newborn rats is mediated through neuronal death, apoptosis, astrogliosis and microglial activation. In this double-hit model, treatment with HT does not ameliorate these changes.

Respiratory sinus arrhythmia stabilizes mean arterial blood pressure at high-frequency interval in healthy humans.

PURPOSE: Arterial blood pressure variations are an independent risk factor for end organ failure. Respiratory sinus arrhythmia (RSA) is a sign of a healthy cardiovascular system. However, whether RSA counteracts arterial blood pressure variations during the respiratory cycle remains controversial. We restricted normal RSA with non-invasive intermittent positive pressure ventilation (IPPV) to test the hypothesis that RSA normally functions to stabilize mean arterial blood pressure. METHODS: Ten young volunteers were investigated during metronome-paced breathing and IPPV. Heart rate (ECG), mean arterial blood pressure and left stroke volume (finger arterial pressure curve) and right stroke volume (pulsed ultrasound Doppler) were recorded, while systemic and pulmonary blood flow were calculated beat-by-beat. Respiratory variations (high-frequency power, 0.15-0.40 Hz) in cardiovascular variables were estimated by spectral analysis. Phase angles and correlation were calculated by cross-spectral analysis. RESULTS: The magnitude of RSA was reduced from 4.9 bpm(2) (95% CI 3.0, 6.2) during metronome breathing to 2.8 bpm(2) (95% CI 1.1, 5.0) during IPPV (p = 0.03). Variations in mean arterial blood pressure were greater (2.3 mmHg(2) (95% CI 1.4, 3.9) during IPPV than during metronome breathing (1.0 mmHg(2) [95% CI 0.7, 1.3]) (p = 0.014). Respiratory variations in right and left stroke volumes were inversely related in the respiratory cycle during both metronome breathing and IPPV. CONCLUSIONS: RSA magnitude is lower and mean arterial blood pressure variability is greater during IPPV than during metronome breathing. We conclude that in healthy humans, RSA stabilizes mean arterial blood pressure at respiratory frequency.

Neither xenon nor fentanyl induces neuroapoptosis in the newborn pig brain.

BACKGROUND: Some inhalation anesthetics increase apoptotic cell death in the developing brain. Xenon, an inhalation anesthetic, increases neuroprotection when combined with therapeutic hypothermia after hypoxic-ischemic brain injury in newborn animals. The authors, therefore, examined whether there was any neuroapoptotic effect of breathing 50% xenon with continuous fentanyl sedation for 24 h at normothermia or hypothermia on newborn pigs. METHODS: Twenty-six healthy pigs (<24-h old) were randomized into four groups: (1) 24 h of 50% inhaled xenon with fentanyl at hypothermia (Trec = 33.5 °C), (2) 24 h of 50% inhaled xenon with fentanyl at normothermia (Trec = 38.5 °C), (3) 24 h of fentanyl at normothermia, or (4) nonventilated juvenile controls at normothermia. Five additional nonrandomized pigs inhaled 2% isoflurane at normothermia for 24 h to verify any proapoptotic effect of inhalation anesthetics in our model. Pathological cells were morphologically assessed in cortex, putamen, hippocampus, thalamus, and white matter. To quantify the findings, immunostained cells (caspase-3 and terminal deoxynucleotidyl transferase-mediated deoxyuridine-triphosphate nick-end labeling) were counted in the same brain regions. RESULTS: For groups (1) to (4), the total number of apoptotic cells was less than 5 per brain region, representing normal developmental neuroapoptosis. After immunostaining and cell counting, regression analysis showed that neither 50% xenon with fentanyl nor fentanyl alone increased neuroapoptosis. Isoflurane caused on average a 5- to 10-fold increase of immunostained cells. CONCLUSION: At normothermia or hypothermia, neither 24 h of inhaled 50% xenon with fentanyl sedation nor fentanyl alone induces neuroapoptosis in the neonatal pig brain. Breathing 2% isoflurane increases neuroapoptosis in neonatal pigs.

Comparing the efficacy in reducing brain injury of different neuroprotective agents following neonatal hypoxia-ischemia in newborn rats: a multi-drug randomized controlled screening trial.

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.

Lipids revert inert Abeta amyloid fibrils to neurotoxic protofibrils that affect learning in mice.

Although soluble oligomeric and protofibrillar assemblies of Abeta-amyloid peptide cause synaptotoxicity and potentially contribute to Alzheimer's disease (AD), the role of mature Abeta-fibrils in the amyloid plaques remains controversial. A widely held view in the field suggests that the fibrillization reaction proceeds 'forward' in a near-irreversible manner from the monomeric Abeta peptide through toxic protofibrillar intermediates, which subsequently mature into biologically inert amyloid fibrils that are found in plaques. Here, we show that natural lipids destabilize and rapidly resolubilize mature Abeta amyloid fibers. Interestingly, the equilibrium is not reversed toward monomeric Abeta but rather toward soluble amyloid protofibrils. We characterized these 'backward' Abeta protofibrils generated from mature Abeta fibers and compared them with previously identified 'forward' Abeta protofibrils obtained from the aggregation of fresh Abeta monomers. We find that backward protofibrils are biochemically and biophysically very similar to forward protofibrils: they consist of a wide range of molecular masses, are toxic to primary neurons and cause memory impairment and tau phosphorylation in mouse. In addition, they diffuse rapidly through the brain into areas relevant to AD. Our findings imply that amyloid plaques are potentially major sources of soluble toxic Abeta-aggregates that could readily be activated by exposure to biological lipids.

Impact of Hypoxia-Ischemia on Neurogenesis and Structural and Functional Outcomes in a Mild-Moderate Neonatal Hypoxia-Ischemia Brain Injury Model.

Hypoxic-ischemic encephalopathy (HIE) is a common type of brain injury caused by a lack of oxygen and blood flow to the brain during the perinatal period. The incidence of HIE is approximately 2−3 cases per 1000 live births in high-income settings; while in low- and middle-income countries, the incidence is 3−10-fold higher. Therapeutic hypothermia (TH) is the current standard treatment for neonates affected by moderate−severe HIE. However, more than 50% of all infants with suspected HIE have mild encephalopathy, and these infants are not treated with TH because of their lower risk of adverse outcomes. Despite this, several analyses of pooled data provide increasing evidence that infants who initially have mild encephalopathy may present signs of more significant brain injury later in life. The purpose of this study was to expand our knowledge about the effect of mild−moderate hypoxia-ischemia (HI) at the cellular, structural, and functional levels. An established rat model of mild−moderate HI was used, where postnatal day (P) 7 rats were exposed to unilateral permanent occlusion of the left carotid artery and 90 min of 8% hypoxia, followed by TH or normothermia (NT) treatment. The extent of injury was assessed using histology (P14 and P42) and MRI (P11 and P32), as well as with short-term and long-term behavioral tests. Neurogenesis was assessed by BrdU staining. We showed that mild−moderate HI leads to a progressive loss of brain tissue, pathological changes in MRI scans, as well as an impairment of long-term motor function. At P14, the median area loss assessed by histology for HI animals was 20% (p < 0.05), corresponding to mild−moderate brain injury, increasing to 55% (p < 0.05) at P42. The data assessed by MRI corroborated our results. HI led to a decrease in neurogenesis, especially in the hippocampus and the lateral ventricle at early time points, with a delayed partial recovery. TH was not neuroprotective at early time points following mild−moderate HI, but prevented the increase in brain damage over time. Additionally, rats treated with TH showed better long-term motor function. Altogether, our results bring more light to the understanding of pathophysiology following mild-moderate HI. We showed that, in the context of mild-moderate HI, TH failed to be significantly neuroprotective. However, animals treated with TH showed a significant improvement in motor, but not cognitive long-term function. These results are in line with what is observed in some cases where neonates with mild HIE are at risk of neurodevelopmental deficits in infancy or childhood. Whether TH should be used as a preventive treatment to reduce adverse outcomes in mild-HIE remains of active interest, and more research has to be carried out in order to address this question.

Temporal Characterization of Microglia-Associated Pro- and Anti-Inflammatory Genes in a Neonatal Inflammation-Sensitized Hypoxic-Ischemic Brain Injury Model.

Hypoxic-ischemic encephalopathy (HIE) mainly affects preterm and term newborns, leading to a high risk of brain damage. Coexisting infection/inflammation and birth asphyxia are key factors associated with intracerebral increase of proinflammatory cytokines linked to HIE. Microglia are key mediators of inflammation during perinatal brain injury, characterized by their phenotypic plasticity, which may facilitate their participation in both the progression and resolution of injury-induced inflammation. The purpose of this study was to investigate the temporal expression of genes associated with pro- and anti-inflammatory cytokines as well as the nucleotide-binding domain, leucine-rich repeat protein (NLRP-3) inflammasome from microglia cells. For this purpose, we used our established neonatal rat model of inflammation-sensitized hypoxic-ischemic (HI) brain injury in seven-day-old rats. We assessed gene expression profiles of 11 cytokines and for NLRP-3 using real-time PCR from sorted CD11b/c microglia of brain samples at different time points (3.5 h after LPS injection and 0, 5, 24, 48, and 72 hours post HI) following different treatments: vehicle, E. coli lipopolysaccharide (LPS), vehicle/HI, and LPS/HI. Our results showed that microglia are early key mediators of the inflammatory response and exacerbate the inflammatory response following HI, polarizing into a predominant proinflammatory M1 phenotype in the early hours post HI. The brains only exposed to HI showed a delay in the expression of proinflammatory cytokines. We also demonstrated that NLRP-3 plays a role in the inflammatory resolution with a high expression after HI insult. The combination of both, a preinfection/inflammation condition and hypoxia-ischemia, resulted in a higher proinflammatory cytokine storm, highlighting the significant contribution of acute inflammation sensitizing prior to a hypoxic insult on the severity of perinatal brain damage.

Variability and sex-dependence of hypothermic neuroprotection in a rat model of neonatal hypoxic-ischaemic brain injury: a single laboratory meta-analysis.

Therapeutic hypothermia (HT) is standard care for term infants with hypoxic-ischaemic (HI) encephalopathy. However, the efficacy of HT in preclinical models, such as the Vannucci model of unilateral HI in the newborn rat, is often greater than that reported from clinical trials. Here, we report a meta-analysis of data from every experiment in a single laboratory, including pilot data, examining the effect of HT in the Vannucci model. Across 21 experiments using 106 litters, median (95% CI) hemispheric area loss was 50.1% (46.0-51.9%; n = 305) in the normothermia group, and 41.3% (35.1-44.9%; n = 317) in the HT group, with a bimodal injury distribution. Median neuroprotection by HT was 17.6% (6.8-28.3%), including in severe injury, but was highly-variable across experiments. Neuroprotection was significant in females (p < 0.001), with a non-significant benefit in males (p = 0.07). Animals representing the median injury in each group within each litter (n = 277, 44.5%) were also analysed using formal neuropathology, which showed neuroprotection by HT throughout the brain, particularly in females. Our results suggest an inherent variability and sex-dependence of the neuroprotective response to HT, with the majority of studies in the Vannucci model vastly underpowered to detect true treatment effects due to the distribution of injury.

Neonatal Systemic Inflammation Induces Inflammatory Reactions and Brain Apoptosis in a Pathogen-Specific Manner.

BACKGROUND: After neonatal asphyxia, therapeutic hypothermia (HT) is the only proven treatment option. Although established as a neuroprotective therapy, benefit from HT has been questioned when infection is a comorbidity to hypoxic-ischaemic (HI) brain injury. Gram-negative and gram-positive species activate the immune system through different pathogen recognition receptors and subsequent immunological systems. In rodent models, gram-negative (lipopolysaccharide [LPS]) and gram-positive (PAM3CSK4 [PAM]) inflammation similarly increase neuronal vulnerability to HI. Interestingly, while LPS pre-sensitisation negates the neuroprotective effect of HT, HT is highly beneficial after PAM-sensitised HI brain injury. OBJECTIVE: We aimed to examine whether systemic gram-positive or gram-negative inflammatory sensitisation affects juvenile rat pups per se, without an HI insult. METHODS: Neonatal 7-day-old rats (n = 215) received intraperitoneal injections of vehicle (0.9% NaCl), LPS (0.1 mg/kg), or PAM (1 mg/kg). Core temperature and weight gain were monitored. Brain cytokine expression (IL-6, IL-1ß, TNF-α, and IL-10, via PCR), apoptosis (cleaved caspase 3, via Western blots), and microglial activation (Iba1, via immunohistochemistry) were examined. RESULTS: LPS induced an immediate drop in core temperature followed by poor weight gain, none of which were seen after PAM. Furthermore, LPS induced brain apoptosis, while PAM did not. The magnitude and temporal profile of brain cytokine expression differed between LPS- and PAM-injected animals. CONCLUSION: These findings reveal sepsis-like conditions and neuroinflammation specific to the inflammatory stimulus (gram-positive vs. gram-negative) in the neonatal rat. They emphasise the importance of pre-clinical models being pathogen dependent, and should always be carefully tailored to their clinical scenario.

Xenon Combined with Therapeutic Hypothermia Is Not Neuroprotective after Severe Hypoxia-Ischemia in Neonatal Rats.

BACKGROUND: Therapeutic hypothermia (TH) is standard treatment following perinatal asphyxia in newborn infants. Experimentally, TH is neuroprotective after moderate hypoxia-ischemia (HI) in seven-day-old (P7) rats. However, TH is not neuroprotective after severe HI. After a moderate HI insult in newborn brain injury models, the anesthetic gas xenon (Xe) doubles TH neuroprotection. The aim of this study was to examine whether combining Xe and TH is neuroprotective as applied in a P7 rat model of severe HI. DESIGN/METHODS: 120 P7 rat pups underwent a severe HI insult; unilateral carotid artery ligation followed by hypoxia (8% O2 for 150min at experimental normothermia (NT-37: Trectal 37°C). Surviving pups were randomised to immediate NT-37 for 5h (n = 36), immediate TH-32: Trectal 32°C for 5h (n = 25) or immediate TH-32 plus 50% inhaled Xe for 5h (n = 24). Pups were sacrificed after one week of survival. Relative area loss of the ligated hemisphere was measured, and neurons in the subventricular zone of this injured hemisphere were counted, to quantify brain damage. RESULTS: Following the HI insult, median (interquartile range, IQR) hemispheric brain area loss was similar in all groups: 63.5% (55.5-75.0) for NT-37 group, 65.0% (57.0-65.0) for TH-32 group, and 66.5% (59.0-72.0) for TH-32+Xe50% group (not significant). Correspondingly, there was no difference in neuronal cell count (NeuN marker) in the subventricular zone across the three treatment groups. CONCLUSIONS: Immediate therapeutic hypothermia with or without additional 50% inhaled Xe, does not provide neuroprotection one week after severe HI brain injury in the P7 neonatal rat. This model aims to mimic the clinical situation in severely asphyxiated neonates and treatment these newborns remains an ongoing challenge.

Monitoring of cerebral blood flow during hypoxia-ischemia and resuscitation in the neonatal rat using laser speckle imaging.

Neonatal hypoxic-ischemic encephalopathy (HIE) is associated with alterations in cerebral blood flow (CBF) as a result of perinatal asphyxia. The extent to whichCBFchanges contribute to injury, and whether treatments that ameliorate these changes might be neuroprotective, is still unknown. Higher throughput techniques to monitorCBFchanges in rodent models ofHIEcan help elucidate the underlying pathophysiology. We developed a laser speckle imaging (LSI) technique to continuously monitorCBFin six postnatal-day 10 (P10) rats simultaneously before, during, and after unilateral hypoxia-ischemia (HI, ligation of the left carotid artery followed by hypoxia in 8% oxygen). After ligation,CBFto the ligated side fell by 30% compared to the unligated side (P < 0.0001). Hypoxia induced a bilateral 55% reduction inCBF, which was partially restored by resuscitation. Compared to resuscitation in air, resuscitation in 100% oxygen increasedCBFto the ligated side by 45% (P = 0.033). Individual variability inCBFresponse to hypoxia between animals accounted for up to 24% of the variability in hemispheric area loss to the ligated side. In both P10 and P7 models of unilateralHI, resuscitation in 100% oxygen did not affect hemispheric area loss, or hippocampalCA1 pyramidal neuron counts, after 1-week survival. ContinuousCBFmonitoring usingLSIin multiple rodents simultaneously can screen potential treatment modalities that affectCBF, and provide insight into the pathophysiology ofHI.

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

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 infants with severe encephalopathy. In a neonatal rat model of unilateral hypoxia-ischaemia (HI), the effect of five different HT temperatures was investigated after either moderate or severe injury. At postnatal-day seven, rat pups underwent moderate or severe HI followed by 5 h at normothermia (37 °C), or one of five HT temperatures: 33.5 °C, 32 °C, 30 °C, 26 °C, and 18 °C. One week after treatment, neuropathological analysis of hemispheric and hippocampal area loss, and CA1 hippocampal pyramidal neuron count, was performed. After moderate injury, a significant reduction in hemispheric and hippocampal loss on the injured side, and preservation of CA1 pyramidal neurons, was seen in the 33.5 °C, 32 °C, and 30 °C groups. Cooling below 33.5 °C did not provide additional neuroprotection. Regardless of treatment temperature, HT was not neuroprotective in the severe HI model. Based on these findings, and previous experience translating preclinical studies into clinical application, we propose that milder cooling should be considered for future clinical trials.

Validation of a neuropathology score using quantitative methods to evaluate brain injury in a pig model of hypoxia ischaemia.

BACKGROUND: Neuropathological examination is the classic outcome measure in experimental studies of newborn brain injury to evaluate novel therapies. We have used a graded neuropathology score in an established global model of perinatal hypoxic-ischaemic (HI) injury. We wished to validate the score using cell counting in our model. NEW METHOD: 32 newborn pigs underwent a 45 min global HI insult then maintained at normothermia (NT, rectal temperature, Trectal 38.5 °C) for 72 h or mild total body hypothermia (HT, Trectal 37.0 °C) combined with selective head cooling for 48 h and subsequently maintained at NT for 24h before brain perfusion fixation. A perinatal pathologist scored haematoxylin and eosin stained 6 µm histological sections for injury in the hippocampus and basal ganglia on a 9-step scale (0.0=no injury, 4.0=>75% injury). We counted the number of healthy neurons in the hippocampus CA1 region and putamen using morphological criteria in eight random, non-overlapping fields from representative sections. RESULTS: Healthy neuronal cell density correlated with neuropathology score in the hippocampus CA1 (r = -0.74) and in the putamen (r = -0.75) and both measures detected a difference between groups. The correlation coefficients were better for the NT compared to the HT group in both the hippocampus (r = -0.87 vs. -0.53) and putamen (r = -0.77 vs. -0.54). COMPARISON WITH EXISTING METHOD: We have validated a histological neuropathological scoring system in our model of perinatal HI by showing correlation between neuronal cell count and estimated injury. CONCLUSIONS: Our neuropathology score is a valid method to assess brain injury with good reproducibility and sensitivity.

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

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 4h 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.

ADAM10, the rate-limiting protease of regulated intramembrane proteolysis of Notch and other proteins, is processed by ADAMS-9, ADAMS-15, and the gamma-secretase.

ADAM10 is involved in the proteolytic processing and shedding of proteins such as the amyloid precursor protein (APP), cadherins, and the Notch receptors, thereby initiating the regulated intramembrane proteolysis (RIP) of these proteins. Here, we demonstrate that the sheddase ADAM10 is also subject to RIP. We identify ADAM9 and -15 as the proteases responsible for releasing the ADAM10 ectodomain, and Presenilin/gamma-Secretase as the protease responsible for the release of the ADAM10 intracellular domain (ICD). This domain then translocates to the nucleus and localizes to nuclear speckles, thought to be involved in gene regulation. Thus, ADAM10 performs a dual role in cells, as a metalloprotease when it is membrane-bound, and as a potential signaling protein once cleaved by ADAM9/15 and the gamma-Secretase.

Glu(332) in the Nicastrin ectodomain is essential for gamma-secretase complex maturation but not for its activity.

The gamma-secretase complex is responsible for the proteolysis of integral membrane proteins. Nicastrin has been proposed to operate as the substrate receptor of the complex with the glutamate 332 (Glu(333) in human) serving as the anionic binding site for the alpha-amino-terminal group of substrates. The putative binding site is located within the aminopeptidase-like domain of Nicastrin. The Glu(332) is proposed to function as the counterpart of the exopeptidase Glu located in the active site of these peptidases. Although Glu(332) could bind the alpha-amino-terminal group of substrates, we hypothesized, in analogy with M28-aminopeptidases, that other residues in the putative binding site of Nicastrin should participate in the interaction as well. Surprisingly, mutagenesis of these residues affected the in vivo processing of APP and Notch substrates only weakly. In addition, the E332Q mutation, which completely abolishes the anionic alpha-amino-terminal binding function, remained fully active. When we introduced the previously characterized E332A mutation, we found strongly decreased gamma-secretase complex levels, but the remaining complex appeared as active as the wild-type complex. We confirmed in two independent in vitro assays that the specific enzymatic activity of the E332A mutant was comparable with that of the wild-type complex. Thus, Glu(332) crucially affects complex maturation rather than substrate recognition. Moreover other Nicastrin mutants, designed to either impede or alter substantially the putative binding pocket, affected only marginally gamma-secretase activity. Consequently, these studies indicate that the main role of the Glu(332) is in the maturation and assembly of gamma-secretase rather than in the recognition of the substrates.