Association of diastolic blood pressure with coronary perfusion pressure during resuscitation in pediatric swine.

BACKGROUND: Diastolic blood pressure (DBP) is suggested as a surrogate for coronary perfusion pressure (CPP) during cardiopulmonary resuscitation. We examined the correlation between DBP and CPP and hypothesized that both would be associated with survival in a pediatric swine model of asphyxial cardiac arrest. METHODS: We performed a retrospective, secondary analysis of 102 pediatric swine resuscitations. DBP and CPP were recorded every 30 s during resuscitation. Values were compared between survivors and non-survivors. RESULTS: DBP mirrored CPP in survivors and non-survivors throughout resuscitation and both were associated with survival. Improvements in DBP and CPP after the first epinephrine administration were greater in survivors (DBP: 25.1 ± 3.0 vs. 5.4 ± 0.8 mmHg, p < 0.01; CPP: 24.9 ± 3.2 vs. 4.8 ± 0.9 mmHg, p < 0.01). DBP and CPP after epinephrine administration were highly predictive of survival, with an area under the curve of 0.95 (0.89-1.00) for DBP and 0.90 (0.81-0.99) for CPP. The optimal threshold for DBP was 22.5 mmHg, whereas that for CPP was 14.5 mmHg. CONCLUSIONS: DBP and CPP were associated with survival throughout resuscitation, and the response of both to the first epinephrine administration was highly predictive of survival in this model. Clinically, the availability of DBP makes it useful as a target for physiologic feedback during resuscitation. IMPACT: Diastolic blood pressure (DBP) mirrored coronary perfusion pressure (CPP) throughout prolonged resuscitation in a pediatric model of asphyxial cardiac arrest. Mean DBP and CPP were significantly greater in survivors than in non-survivors both before and after administration of epinephrine. The response of both DBP and CPP to the first dose of epinephrine was highly predictive of return of spontaneous circulation. Given the clinical availability of DBP, these findings support its use as a surrogate for CPP to guide high-quality cardiopulmonary resuscitation in this pediatric swine model.

Biological and Procedural Predictors of Outcome in the Stroke Preclinical Assessment Network (SPAN) Trial.

BACKGROUND: The SPAN trial (Stroke Preclinical Assessment Network) is the largest preclinical study testing acute stroke interventions in experimental focal cerebral ischemia using endovascular filament middle cerebral artery occlusion (MCAo). Besides testing interventions against controls, the prospective design captured numerous biological and procedural variables, highlighting the enormous heterogeneity introduced by the multicenter structure that might influence stroke outcomes. Here, we leveraged the unprecedented sample size achieved by the SPAN trial and the prospective design to identify the biological and procedural variables that affect experimental stroke outcomes in transient endovascular filament MCAo. METHODS: The study cohort included all mice enrolled and randomized in the SPAN trial (N=1789). Mice were subjected to 60-minute MCAo and followed for a month. Thirteen biological and procedural independent variables and 4 functional (weight loss and 4-point neuroscore on days 1 and 2, corner test on days 7 and 28, and mortality) and 3 tissue (day 2, magnetic resonance imaging infarct volumes and swelling; day 30, magnetic resonance imaging tissue loss) outcome variables were prospectively captured. Multivariable regression with stepwise elimination was used to identify the predictors and their effect sizes. RESULTS: Older age, active circadian stage at MCAo, and thinner and longer filament silicone tips predicted higher mortality. Older age, larger body weight, longer anesthesia duration, and longer filament tips predicted worse neuroscores, while high-fat diet and blood flow monitoring predicted milder neuroscores. Older age and a high-fat diet predicted worse corner test performance. While shorter filament tips predicted more ipsiversive turning, longer filament tips appeared to predict contraversive turning. Age, sex, and weight interacted when predicting the infarct volume. Older age was associated with smaller infarcts on day 2 magnetic resonance imaging, especially in animals with larger body weights; this association was most conspicuous in females. High-fat diet also predicted smaller infarcts. In contrast, the use of cerebral blood flow monitoring and more severe cerebral blood flow drop during MCAo, longer anesthesia, and longer filament tips all predicted larger infarcts. Bivariate analyses among the dependent variables highlighted a disconnect between tissue and functional outcomes. CONCLUSIONS: Our analyses identified variables affecting endovascular filament MCAo outcome, an experimental stroke model used worldwide. Multiple regression refuted some commonly reported predictors and revealed previously unrecognized associations. Given the multicenter prospective design that represents a sampling of real-world conditions, the degree of heterogeneity mimicking clinical trials, the large number of predictors adjusted for in the multivariable model, and the large sample size, we think this is the most definitive analysis of the predictors of preclinical stroke outcome to date. Future multicenter experimental stroke trials should standardize or at least ensure a balanced representation of the biological and procedural variables identified herein as potential confounders.

Neuroprotection provided by hypothermia initiated with high transnasal flow with ambient air in a model of pediatric cardiac arrest.

Clinical trials of hypothermia after pediatric cardiac arrest (CA) have not seen robust improvement in functional outcome, possibly because of the long delay in achieving target temperature. Previous work in infant piglets showed that high nasal airflow, which induces evaporative cooling in the nasal mucosa, reduced regional brain temperature uniformly in half the time needed to reduce body temperature. Here, we evaluated whether initiation of hypothermia with high transnasal airflow provides neuroprotection without adverse effects in the setting of asphyxic CA. Anesthetized piglets underwent sham-operated procedures (n = 7) or asphyxic CA with normothermic recovery (38.5°C; n = 9) or hypothermia initiated by surface cooling at 10 (n = 8) or 120 (n = 7) min or transnasal cooling initiated at 10 (n = 7) or 120 (n = 7) min after resuscitation. Hypothermia was sustained at 34°C with surface cooling until 20 h followed by 6 h of rewarming. At 4 days of recovery, significant neuronal loss occurred in putamen and sensorimotor cortex. Transnasal cooling initiated at 10 min significantly rescued the number of viable neurons in putamen, whereas levels in putamen in other hypothermic groups remained less than sham levels. In sensorimotor cortex, neuronal viability in the four hypothermic groups was not significantly different from the sham group. These results demonstrate that early initiation of high transnasal airflow in a pediatric CA model is effective in protecting vulnerable brain regions. Because of its simplicity, portability, and low cost, transnasal cooling potentially could be deployed in the field or emergency room for early initiation of brain cooling after pediatric CA.NEW & NOTEWORTHY The onset of therapeutic hypothermia after cardiac resuscitation is often delayed, leading to incomplete neuroprotection. In an infant swine model of asphyxic cardiac arrest, initiation of high transnasal airflow to maximize nasal evaporative cooling produced hypothermia sufficient to provide neuroprotection that was not inferior to body surface cooling. Because of its simplicity and portability, this technique may be of use in the field or emergency room for rapid brain cooling in pediatric cardiac arrest victims.

Soluble PD-L1 reprograms blood monocytes to prevent cerebral edema and facilitate recovery after ischemic stroke.

Acute cerebral ischemia triggers a profound inflammatory response. While macrophages polarized to an M2-like phenotype clear debris and facilitate tissue repair, aberrant or prolonged macrophage activation is counterproductive to recovery. The inhibitory immune checkpoint Programmed Cell Death Protein 1 (PD-1) is upregulated on macrophage precursors (monocytes) in the blood after acute cerebrovascular injury. To investigate the therapeutic potential of PD-1 activation, we immunophenotyped circulating monocytes from patients and found that PD-1 expression was upregulated in the acute period after stroke. Murine studies using a temporary middle cerebral artery (MCA) occlusion (MCAO) model showed that intraperitoneal administration of soluble Programmed Death Ligand-1 (sPD-L1) significantly decreased brain edema and improved overall survival. Mice receiving sPD-L1 also had higher performance scores short-term, and more closely resembled sham animals on assessments of long-term functional recovery. These clinical and radiographic benefits were abrogated in global and myeloid-specific PD-1 knockout animals, confirming PD-1+ monocytes as the therapeutic target of sPD-L1. Single-cell RNA sequencing revealed that treatment skewed monocyte maturation to a non-classical Ly6Clo, CD43hi, PD-L1+ phenotype. These data support peripheral activation of PD-1 on inflammatory monocytes as a therapeutic strategy to treat neuroinflammation after acute ischemic stroke.

Hypothermic Protection in Neocortex Is Topographic and Laminar, Seizure Unmitigating, and Partially Rescues Neurons Depleted of RNA Splicing Protein Rbfox3/NeuN in Neonatal Hypoxic-Ischemic Male Piglets.

The effects of hypothermia on neonatal encephalopathy may vary topographically and cytopathologically in the neocortex with manifestations potentially influenced by seizures that alter the severity, distribution, and type of neuropathology. We developed a neonatal piglet survival model of hypoxic-ischemic (HI) encephalopathy and hypothermia (HT) with continuous electroencephalography (cEEG) for seizures. Neonatal male piglets received HI-normothermia (NT), HI-HT, sham-NT, or sham-HT treatments. Randomized unmedicated sham and HI piglets underwent cEEG during recovery. Survival was 2-7 days. Normal and pathological neurons were counted in different neocortical areas, identified by cytoarchitecture and connectomics, using hematoxylin and eosin staining and immunohistochemistry for RNA-binding FOX-1 homolog 3 (Rbfox3/NeuN). Seizure burden was determined. HI-NT piglets had a reduced normal/total neuron ratio and increased ischemic-necrotic/total neuron ratio relative to sham-NT and sham-HT piglets with differing severities in the anterior and posterior motor, somatosensory, and frontal cortices. Neocortical neuropathology was attenuated by HT. HT protection was prominent in layer III of the inferior parietal cortex. Rbfox3 immunoreactivity distinguished cortical neurons as: Rbfox3-positive/normal, Rbfox3-positive/ischemic-necrotic, and Rbfox3-depleted. HI piglets had an increased Rbfox3-depleted/total neuron ratio in layers II and III compared to sham-NT piglets. Neuronal Rbfox3 depletion was partly rescued by HT. Seizure burdens in HI-NT and HI-HT piglets were similar. We conclude that the neonatal HI piglet neocortex has: (1) suprasylvian vulnerability to HI and seizures; (2) a limited neuronal cytopathological repertoire in functionally different regions that engages protective mechanisms with HT; (3) higher seizure burden, insensitive to HT, that is correlated with more panlaminar ischemic-necrotic neurons in the somatosensory cortex; and (4) pathological RNA splicing protein nuclear depletion that is sensitive to HT. This work demonstrates that HT protection of the neocortex in neonatal HI is topographic and laminar, seizure unmitigating, and restores neuronal depletion of RNA splicing factor.

A multi-laboratory preclinical trial in rodents to assess treatment candidates for acute ischemic stroke.

Human diseases may be modeled in animals to allow preclinical assessment of putative new clinical interventions. Recent, highly publicized failures of large clinical trials called into question the rigor, design, and value of preclinical assessment. We established the Stroke Preclinical Assessment Network (SPAN) to design and implement a randomized, controlled, blinded, multi-laboratory trial for the rigorous assessment of candidate stroke treatments combined with intravascular thrombectomy. Efficacy and futility boundaries in a multi-arm multi-stage statistical design aimed to exclude from further study highly effective or futile interventions after each of four sequential stages. Six independent research laboratories performed a standard focal cerebral ischemic insult in five animal models that included equal numbers of males and females: young mice, young rats, aging mice, mice with diet-induced obesity, and spontaneously hypertensive rats. The laboratories adhered to a common protocol and efficiently enrolled 2615 animals with full data completion and comprehensive animal tracking. SPAN successfully implemented treatment masking, randomization, prerandomization inclusion and exclusion criteria, and blinded assessment of outcomes. The SPAN design and infrastructure provide an effective approach that could be used in similar preclinical, multi-laboratory studies in other disease areas and should help improve reproducibility in translational science.

Improving outcomes in intracerebral hemorrhage through microglia/macrophage-targeted IL-10 delivery with phosphatidylserine liposomes.

Intracerebral hemorrhage (ICH) remains the most lethal type of stroke, and effective clinical therapies that can speed up hematoma resolution after ICH are still lacking. While the beneficial effects of IL-10 on ICH recovery have been demonstrated, the clinical translation of IL-10 requires effective delivery methods by which sufficient IL-10 can be delivered to ICH-affected regions in the brain. Here we report the use of a phosphatidylserine (PS) liposome (PSL)-based nanoparticle system for microglia/macrophage-targeted delivery of IL-10 in ICH. We first prepared IL-10-conjugated PSL (PSL-IL10) and characterized their immunomodulating effects in vitro. Then we evaluated the therapeutic effects, including hematoma absorption, short-term outcomes, and neuroinflammation, of intranasally administered PSL-IL10 (3 µg IL-10 per mouse, 2 h post-ICH) in a collagenase-induced ICH mouse model. We also isolated microglia/macrophages from the mouse brains with ICH to analyze their morphology, phagocytosis ability, and polarization. Our study reveals that, 1) PSL-IL10 treatment resulted in significantly improved outcomes and accelerated hematoma resolution in the acute phase of ICH; 2) PSL-IL10 inhibited glial activation and down-regulated pro-inflammatory cytokine production; 3) PSL-IL10 induced Iba1+ cells with a stronger phagocytosis ability; 4) PSL-IL10 activated STAT3 and upregulated CD36 expression in microglia/macrophage. These findings collectively show that PSL-IL10 is a promising nanotherapeutic for effectively ameliorating ICH.

Rapid, selective and homogeneous brain cooling with transnasal flow of ambient air for pediatric resuscitation.

Neurologic outcome from out-of-hospital pediatric cardiac arrest remains poor. Although therapeutic hypothermia has been attempted in this patient population, a beneficial effect has yet to be demonstrated, possibly because of the delay in achieving target temperature. To minimize this delay, we developed a simple technique of transnasal cooling. Air at ambient temperature is passed through standard nasal cannula with an open mouth to produce evaporative cooling of the nasal passages. We evaluated efficacy of brain cooling with different airflows in different size piglets. Brain temperature decreased by 3°C within 25 minutes with nasal airflow rates of 16, 32, and 16 L/min in 1.8-, 4-, and 15-kg piglets, respectively, whereas rectal temperature lagged brain temperature. No substantial spatial temperature gradients were seen along the neuroaxis, suggesting that heat transfer is via blood convection. The evaporative cooling did not reduce nasal turbinate blood flow or sagittal sinus oxygenation. The rapid and selective brain cooling indicates a high humidifying capacity of the nasal turbinates is present early in life. Because of its simplicity, portability, and low cost, transnasal cooling potentially could be deployed in the field for early initiation of brain cooling prior to maintenance with standard surface cooling after pediatric cardiac arrest.

Polynitroxylated PEGylated hemoglobin protects pig brain neocortical gray and white matter after traumatic brain injury and hemorrhagic shock.

Polynitroxylated PEGylated hemoglobin (PNPH, aka SanFlow) possesses superoxide dismutase/catalase mimetic activities that may directly protect the brain from oxidative stress. Stabilization of PNPH with bound carbon monoxide prevents methemoglobin formation during storage and permits it to serve as an anti-inflammatory carbon monoxide donor. We determined whether small volume transfusion of hyperoncotic PNPH is neuroprotective in a porcine model of traumatic brain injury (TBI) with and without accompanying hemorrhagic shock (HS). TBI was produced by controlled cortical impact over the frontal lobe of anesthetized juvenile pigs. Hemorrhagic shock was induced starting 5 min after TBI by 30 ml/kg blood withdrawal. At 120 min after TBI, pigs were resuscitated with 60 ml/kg lactated Ringer's (LR) or 10 or 20 ml/kg PNPH. Mean arterial pressure recovered to approximately 100 mmHg in all groups. A significant amount of PNPH was retained in the plasma over the first day of recovery. At 4 days of recovery in the LR-resuscitated group, the volume of frontal lobe subcortical white matter ipsilateral to the injury was 26.2 ± 7.6% smaller than homotypic contralateral volume, whereas this white matter loss was only 8.6 ± 12.0% with 20-ml/kg PNPH resuscitation. Amyloid precursor protein punctate accumulation, a marker of axonopathy, increased in ipsilateral subcortical white matter by 132 ± 71% after LR resuscitation, whereas the changes after 10 ml/kg (36 ± 41%) and 20 ml/kg (26 ± 15%) PNPH resuscitation were not significantly different from controls. The number of cortical neuron long dendrites enriched in microtubules (length >50 microns) decreased in neocortex by 41 ± 24% after LR resuscitation but was not significantly changed after PNPH resuscitation. The perilesion microglia density increased by 45 ± 24% after LR resuscitation but was unchanged after 20 ml/kg PNPH resuscitation (4 ± 18%). Furthermore, the number with an activated morphology was attenuated by 30 ± 10%. In TBI pigs without HS followed 2 h later by infusion of 10 ml/kg LR or PNPH, PNPH remained neuroprotective. These results in a gyrencephalic brain show that resuscitation from TBI + HS with PNPH protects neocortical gray matter, including dendritic microstructure, and white matter axons and myelin. This neuroprotective effect persists with TBI alone, indicating brain-targeting benefits independent of blood pressure restoration.

Embolic occlusion of internal carotid artery in conscious rats: Immediate effects of cerebral ischemia.

In most preclinical models of focal ischemic stroke, vascular occlusion is performed under general anesthesia. However, anesthetic agents exert confounding effects on mean arterial blood pressure (MABP), cerebrovascular tone, oxygen demand, and neurotransmitter receptor transduction. Moreover, the majority of studies do not use a blood clot, which more fully models embolic stroke. Here, we developed a blood clot injection model to produce large cerebral artery ischemia in unanesthetized rats. Under isoflurane anesthesia, an indwelling catheter was implanted in the internal carotid artery via a common carotid arteriotomy and preloaded with a 0.38-mm-diameter clot of 1.5, 3, or 6 cm length. After discontinuing anesthesia, the rat was returned to a home cage where it regained normal mobility, grooming, eating activity, and a stable recovery of MABP. One hour later, the clot was injected over a 10-s period and the rats were observed for 24 h. Clot injection produced a brief period of irritability, then 15-20 min of complete inactivity, followed by lethargic activity at 20-40 min, ipsilateral deviation of the head and neck at 1-2 h, and limb weakness and circling at 2-4 h. Neurologic deficits, elevated MABP, infarct volume, and increased hemisphere water content varied directly with clot size. Mortality after 6-cm clot injection (53%) was greater than that after 1.5-cm (10%) or 3-cm (20%) injection. Combined non-survivor groups had the greatest MABP, infarct volume, and water content. Among all groups, the pressor response correlated with infarct volume. The coefficient of variation of infarct volume with the 3-cm clot was less than that in published studies with the filament or standard clot models, and therefore may provide stronger statistical power for stroke translational studies. The more severe outcomes from the 6-cm clot model may be useful for the study of malignant stroke.

Importance of Control Groups for Evaluating Long-Term Behavioral and Cognitive Outcomes of Controlled Cortical Impact in Immature Rats.

Therapies are limited for pediatric traumatic brain injury (TBI), especially for the very young who can experience long-term consequences to learning, memory, and social behavior. Animal models of pediatric TBI have yielded mechanistic insights, but demonstration of clinically relevant long-term behavioral and/or cognitive deficits has been challenging. We characterized short- and long-term outcomes in a controlled cortical impact (CCI) model of pediatric TBI using a panel of tests between 2 weeks and ∼4 months after injury. Male rats with CCI at postnatal Day (PND) 10 were compared with three control groups: Naïve, Anesthesia, and Craniotomy. Motor testing (PND 25-33), novel object recognition (NOR; PND 40-50), and multiple tasks in water maze (WM; PND 65-100) were followed by social interaction tests (PND 120-140). Anesthesia rats performed the same as Naïve rats in all tasks. TBI rats, when compared with Naïve controls, had functional impairments across most tests studied. The most sensitive cognitive processes affected by TBI included those that required fast one-trial learning (NOR, WM), flexibility of acquired memory traces (reversals in WM), response strategies (WM), or recognition memory in the setting of reciprocal social interactions. Both TBI and Craniotomy groups demonstrated increased rates of decision making across several WM tasks, suggesting disinhibition of motor responses. When the TBI group was compared with the Craniotomy group, however, deficits were detected in a limited number of outcomes. The latter included learning speed (WM), cognitive flexibility (WM), and social recognition memory. Notably, effects of craniotomy, when compared with Naïve controls, spanned across multiple tasks, and in some tasks, could reach the effect sizes observed in TBI. These results highlight the importance of appropriate control groups in pediatric CCI models. In addition, the study demonstrates the high sensitivity of comprehensive cognitive testing to detect long-term effects of early-age craniotomy and TBI and provides a template for future testing of experimental therapies.

Amide Proton Transfer-Weighted Magnetic Resonance Imaging for Detecting Severity and Predicting Outcome after Traumatic Brain Injury in Rats.

After traumatic brain injury (TBI), early assessment of secondary injury severity is critically important for estimating prognosis and treatment stratification. Currently, secondary injury severity is difficult to estimate. The objective of this study was to investigate the capacity of non-invasive amide proton transfer-weighted (APTw) magnetic resonance imaging (MRI) techniques to assess TBI injury in different brain regions and predict long-term neurobehavior outcomes. Fifty-five male and female rats were subjected to a controlled cortical impact with one of three different impactor depths to produce different degrees of TBI. Multi-parameter MRI data were acquired on a 4.7-Tesla scanner at 1 h, 1 day, and 3 days. Immunofluorescence staining was used to detect activated microglia at 3 days, and neurobehavioral tests were performed to assess long-term outcomes after 28 days. The APTw signal in the injury core at 1 day correlated with deficits in sensorimotor function, the sucrose preference test (a test for anhedonia), and spatial memory function on the Barnes maze. The APTw signal in the perilesion ipsilateral cortex gradually increased after TBI, and the value at 3 days correlated with microglia density at 3 days and with spatial memory decline and anhedonia at 28 days. The correlation between APTw and activated microglia was also observed in the ipsilateral thalamus, and its correlation to memory deficit and depression was evident in other ipsilateral sites. These results suggest that APTw imaging can be used for detecting secondary injury and as a potential predictor of long-term outcomes from TBI.

Interleukin-10 deficiency aggravates traumatic brain injury in male but not female mice.

The goal of this study was to determine whether deficiency of anti-inflammatory cytokine interleukin-10 (IL-10) affects traumatic brain injury (TBI) outcomes in a sex-dependent manner. Moderate TBI was induced by controlled cortical impact in 8-10 week-old wild-type and IL-10-deficient mice. In wild-type mice, serum IL-10 was significantly increased after TBI in males but not in females. At 4-5 weeks after TBI, sensorimotor function, cognitive function (Y-maze and novel object recognition tests), anxiety-related behavior (light-dark box and open field test), and depression-like behavior (forced swim test) were assessed. IL-10-deficient male mice had larger lesion volumes than did wild-type mice in the early recovery phase and worse performance on sensorimotor tasks, cognitive tests, and anxiety- and depression-related tests in the late recovery phase, whereas female IL-10-deficient mice had lesion volume equivalent to that of wild-type females and worse performance only on sensorimotor tasks. At 3 days after TBI, the number of GFAP- and Iba1-positive cells were augmented in areas in proximity to the injury (cerebral cortex and hippocampus) and in remote functional regions (striatum and amygdala) of IL-10-deficient male, but not female, mice. Moreover, on day 35, significantly fewer NeuN-positive cells were present in cortex, striatum, and amygdala of IL-10-deficient male mice than in wild-type males. This difference was not evident in females. We conclude that IL-10 deficiency aggravates cognitive and emotional recovery from TBI in association with enhanced gliosis and neuronal loss selectively in males, suggesting that recruitment of this cytokine limits damage in a sex-dependent manner.

Comparison of different metrics of cerebral autoregulation in association with major morbidity and mortality after cardiac surgery.

BACKGROUND: Cardiac surgery studies have established the clinical relevance of personalised arterial blood pressure management based on cerebral autoregulation. However, variabilities exist in autoregulation evaluation. We compared the association of several cerebral autoregulation metrics, calculated using different methods, with outcomes after cardiac surgery. METHODS: Autoregulation was measured during cardiac surgery in 240 patients. Mean flow index and cerebral oximetry index were calculated as Pearson's correlations between mean arterial pressure (MAP) and transcranial Doppler blood flow velocity or near-infrared spectroscopy signals. The lower limit of autoregulation and optimal mean arterial pressure were identified using mean flow index and cerebral oximetry index. Regression models were used to examine associations of area under curve and duration of mean arterial pressure below thresholds with stroke, acute kidney injury (AKI), and major morbidity and mortality. RESULTS: Both mean flow index and cerebral oximetry index identified the cerebral lower limit of autoregulation below which MAP was associated with a higher incidence of AKI and major morbidity and mortality. Based on magnitude and significance of the estimates in adjusted models, the area under curve of MAP < lower limit of autoregulation had the strongest association with AKI and major morbidity and mortality. The odds ratio for area under the curve of MAP < lower limit of autoregulation was 1.05 (95% confidence interval, 1.01-1.09), meaning every 1 mm Hg h increase of area under the curve was associated with an average increase in the odds of AKI by 5%. CONCLUSIONS: For cardiac surgery patients, area under curve of MAP < lower limit of autoregulation using mean flow index or cerebral oximetry index had the strongest association with AKI and major morbidity and mortality. Trials are necessary to evaluate this target for MAP management.

Neuroprotection in the Striatum of Hypoxic-Ischemic Piglets by Simultaneous Inhibition of Dopamine D1 and Adenosine A2A Receptors.

INTRODUCTION: Striatal neurons of term newborns are highly vulnerable to hypoxia-ischemia (H-I). In a piglet model of H-I, a dopamine D1 receptor antagonist and an adenosine A2A receptor antagonist alone preferentially protect striatonigral and striatopallidal neurons, respectively. Here, we tested the hypothesis whether the combined treatment with SCH23390, a D1 receptor antagonist, and SCH58261, an A2A receptor antagonist, is more efficacious than individual D1 and A2A receptor antagonist treatment. METHODS: Anesthetized newborn piglets were subjected to sham operation (n = 6) or 40 min of hypoxia and 7 min of airway occlusion. At 5 min of reoxygenation, piglets received the vehicle, SCH23390, SCH58261, or the combined treatment (n = 9 in each group). At 4 days of recovery, the number of viable neurons in the entire putamen was estimated by unbiased stereology. RESULTS: Stereological results showed that sham-operated piglets had an estimated 2.9 × 106 neurons in the putamen, and the number of viable neurons in hypoxic-ischemic piglets was significantly reduced by 80% to 0.6 × 106/putamen. Treatment with SCH23390, SCH58261, and the combination increased the numbers of viable neurons to 1.4 × 106/putamen, 1.4 × 106/putamen, and 2.1 × 106/putamen, respectively. Notably, the combined treatment improved neuroprotection compared to individual therapy. CONCLUSION: We conclude that simultaneous inhibition of dopamine D1 receptors and adenosine A2A receptors saves more neurons than individual treatment in the highly vulnerable putamen of a large-animal neonatal H-I model.

The Stroke Preclinical Assessment Network: Rationale, Design, Feasibility, and Stage 1 Results.

Cerebral ischemia and reperfusion initiate cellular events in brain that lead to neurological disability. Investigating these cellular events provides ample targets for developing new treatments. Despite considerable work, no such therapy has translated into successful stroke treatment. Among other issues-such as incomplete mechanistic knowledge and faulty clinical trial design-a key contributor to prior translational failures may be insufficient scientific rigor during preclinical assessment: nonblinded outcome assessment; missing randomization; inappropriate sample sizes; and preclinical assessments in young male animals that ignore relevant biological variables, such as age, sex, and relevant comorbid diseases. Promising results are rarely replicated in multiple laboratories. We sought to address some of these issues with rigorous assessment of candidate treatments across 6 independent research laboratories. The Stroke Preclinical Assessment Network (SPAN) implements state-of-the-art experimental design to test the hypothesis that rigorous preclinical assessment can successfully reduce or eliminate common sources of bias in choosing treatments for evaluation in clinical studies. SPAN is a randomized, placebo-controlled, blinded, multilaboratory trial using a multi-arm multi-stage protocol to select one or more putative stroke treatments with an implied high likelihood of success in human clinical stroke trials. The first stage of SPAN implemented procedural standardization and experimental rigor. All participating research laboratories performed middle cerebral artery occlusion surgery adhering to a common protocol and rapidly enrolled 913 mice in the first of 4 planned stages with excellent protocol adherence, remarkable data completion and low rates of subject loss. SPAN stage 1 successfully implemented treatment masking, randomization, prerandomization inclusion/exclusion criteria, and blinded assessment to exclude bias. Our data suggest that a large, multilaboratory, preclinical assessment effort to reduce known sources of bias is feasible and practical. Subsequent SPAN stages will evaluate candidate treatments for potential success in future stroke clinical trials using aged animals and animals with comorbid conditions.

Transcranial photoacoustic characterization of neurovascular physiology during early-stage photothrombotic stroke in neonatal pigletsin vivo.

OBJECTIVE: Perinatal ischemic stroke is estimated to occur in 1/2300-1/5000 live births, but early differential diagnosis from global hypoxia-ischemia is often difficult. In this study, we tested the ability of a hand-held transcranial photoacoustic (PA) imaging probe to non-invasively detect a focal photothrombotic stroke (PTS) within 2 h of stroke onset in a gyrencephalic piglet brain. APPROACH: About 17 stroke lesions of approximately 1 cm2area were introduced randomly in anterior or posterior cortex via the light/dye PTS technique in anesthetized neonatal piglets (n= 11). The contralateral non-ischemic region served as control tissue for discrimination contrast for the PA hemoglobin metrics: oxygen saturation, total hemoglobin (tHb), and individual quantities of oxygenated and deoxygenated hemoglobin (HbO2and HbR). MAIN RESULTS: The PA-derived tissue oxygen saturation at 2 h yielded a significant separation between control and affected regions-of-interest (p< 0.0001), which were well matched with 24 h post-stroke cerebral infarction confirmed in the triphenyltetrazolium chloride-stained image. The quantity of HbO2also displayed a significant contrast (p= 0.021), whereas tHb and HbR did not. The analysis on receiver operating characteristic curves and multivariate data analysis also agreed with the results above. SIGNIFICANCE: This study shows that a hand-held transcranial PA neuroimaging device can detect a regional thrombotic stroke in the cerebral cortex of a neonatal piglet. In particular, we conclude that the oxygen saturation metric can be used alone to identify regional stroke lesions. The lack of change in tHb may be related to arbitrary hand-held imaging configuration and/or entrapment of red blood cells within the thrombotic stroke.

Photoacoustic assessment of the fetal brain and placenta as a method of non-invasive antepartum and intrapartum monitoring.

A noninvasive monitor for concurrent evaluation of placental and fetal sagittal sinus sO 2 for both antepartum surveillance at the late 2nd and 3rd trimesters and intrapartum monitoring would be a great advantage over current methods. A PA fetal brain and placental monitor has potential value to rapidly identify the fetus at risk for developing hypoxia and ischemia of a sufficient degree that brain injury or death may develop, which may be prevented by intervention with delivery and other follow-up treatments.

20-HETE Participates in Intracerebral Hemorrhage-Induced Acute Injury by Promoting Cell Ferroptosis.

Intracerebral hemorrhage (ICH) is a highly fatal type of stroke that leads to various types of neuronal death. Recently, ferroptosis, a form of cell death resulting from iron-dependent lipid peroxide accumulation, was observed in a mouse ICH model. N-hydroxy-N'-(4-n-butyl-2-methylphenyl)-formamidine (HET0016), which inhibits synthesis of the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE), has shown a protective effect after ICH. However, the underlying mechanisms of the neuroprotective effect need further investigation. We explored whether 20-HETE participates in ICH-induced ferroptosis ex vivo by using hemoglobin-treated organotypic hippocampal slice cultures (OHSCs) and in vivo by using a collagenase-induced ICH mouse model. Ex vivo, we found that the 20-HETE synthesis inhibitor HET0016 and antagonist 20-6,15-HEDGE reduced hemoglobin-induced cell death, iron deposition, and lipid reactive oxygen species levels in OHSCs. Furthermore, 20-HETE inhibition in OHSCs increased the expression of glutathione peroxidase (GPX) 4, an antioxidant enzyme that serves as a main regulator of ferroptosis. In contrast, exposure of OHSCs to the 20-HETE stable mimetic 20-5,14-HEDGE induced cell death that was significantly inhibited by the ferroptosis inhibitor ferrostatin-1. In vivo, HET0016 treatment ameliorated focal deficits, reduced lesion volume, and decreased iron accumulation around the lesion at day 3 and 7 after ICH. In addition, lipid peroxidation was decreased and expression of GPX4 was increased in the HET0016-treated ICH group. The mitogen-activated protein kinase pathway also was inhibited by HET0016 in vivo. These results indicate that 20-HETE contributes to ICH-induced acute brain injury in part by activating ferroptosis pathways, thereby providing an upstream target for inhibiting ferroptosis.