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) minutes or transnasal cooling initiated at 10 (n=7) or 120 (n=7) minutes after resuscitation. Hypothermia was sustained at 34°C with surface cooling until 20 hours followed by 6 hours of rewarming. At four days of recovery, significant neuronal loss occurred in putamen and sensorimotor cortex. Transnasal cooling initiated at 10 minutes 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.

Targeting the mitochondrial permeability transition pore for neuroprotection in a piglet model of neonatal hypoxic-ischemic encephalopathy.

Neonatal hypoxic-ischemic encephalopathy (HIE) causes significant morbidity despite treatment with therapeutic hypothermia. Mitochondrial dysfunction may drive the mechanisms underlying neuronal cell death, thereby making mitochondria prime targets for neuroprotection. The mitochondrial permeability transition pore (mPTP) is one such target within mitochondria. In adult animal models, mPTP inhibition is neuroprotective. However, evidence for mPTP inhibition in neonatal models of neurologic disease is less certain. We tested the therapeutic efficacy of the mPTP small molecule inhibitor GNX-4728 and examined the developmental presence of brain mPTP proteins for drug targeting in a neonatal piglet model of hypoxic-ischemic brain injury. Male neonatal piglets were randomized to hypoxia-ischemia (HI) or sham procedure with GNX-4728 (15 mg/kg, IV) or vehicle (saline/cyclodextrin/DMSO, IV). GNX-4728 was administered as a single dose within 5 min after resuscitation from bradycardic arrest. Normal, ischemic, and injured neurons were counted in putamen and somatosensory cortex using hematoxylin and eosin staining. In separate neonatal and juvenile pigs, western blots of putamen mitochondrial-enriched fractions were used to evaluate mitochondrial integrity and the presence of mPTP proteins. We found that a single dose of GNX-4728 did not protect putamen and cortical neurons from cell death after HI. However, loss of mitochondrial matrix integrity occurred within 6h after HI, and while mPTP components are present in the neonatal brain their levels were significantly different compared to that of a mature juvenile brain. Thus, the neonatal brain mPTP may not be a good target for current neurotherapeutic drugs that are developed based on adult mitochondria.

Quantitative validation of MRI mapping of cerebral venous oxygenation with direct blood sampling: A graded-O2 study in piglets.

PURPOSE: To validate two neonatal cerebral venous oxygenation (Yv ) MRI techniques, T2 relaxation under phase contrast (TRUPC) and accelerated TRUPC (aTRUPC) MRI, with oxygenation measured with direct blood sampling. METHODS: In vivo experiments were performed on seven healthy newborn piglets. For each piglet, a catheter was placed in the superior sagittal sinus to obtain venous blood samples for blood gas oximetry measurement as a gold standard. During the MRI experiment, three to five venous oxygenation levels were achieved in each piglet by varying inhaled O2 content and breathing rate. Under each condition, Yv values of the superior sagittal sinus measured by TRUPC, aTRUPC, and blood gas oximetry were obtained. The Yv quantification in TRUPC and aTRUPC used a standard bovine blood calibration model. The aTRUPC scan was repeated twice to assess its reproducibility. Agreements among TRUPC Yv , aTRUPC Yv , and blood gas oximetry were evaluated by intraclass correlation coefficient (ICC) and paired Student's t-test. RESULTS: The mean hematocrit was 23.6 ± 6.5% among the piglets. Across all measurements, Yv values were 51.9 ± 21.3%, 54.1 ± 18.8%, and 53.7 ± 19.2% for blood gas oximetry, TRUPC and aTRUPC, respectively, showing no significant difference between any two methods (P > .3). There were good correlations between TRUPC and blood gas Yv (ICC = 0.801; P < .0001), between aTRUPC and blood gas Yv (ICC = 0.809; P < .0001), and between aTRUPC and TRUPC Yv (ICC = 0.887; P < .0001). The coefficient of variation of aTRUPC Yv was 8.1 ± 9.9%. CONCLUSION: The values of Yv measured by TRUPC and aTRUPC were in good agreement with blood gas oximetry. These findings suggest that TRUPC and aTRUPC can provide accurate quantifications of Yv in major cerebral veins.

Multi-Parametric Evaluation of Cerebral Hemodynamics in Neonatal Piglets Using Non-Contrast-Enhanced Magnetic Resonance Imaging Methods.

BACKGROUND: Disruption of brain oxygen delivery and consumption after hypoxic-ischemic injury contributes to neonatal mortality and neurological impairment. Measuring cerebral hemodynamic parameters, including cerebral blood flow (CBF), oxygen extraction fraction (OEF), and cerebral metabolic rate of oxygen (CMRO2 ), is clinically important. PURPOSE: Phase-contrast (PC), velocity-selective arterial spin labeling (VSASL), and T2 -relaxation-under-phase-contrast (TRUPC) are magnetic resonance imaging (MRI) techniques that have shown promising results in assessing cerebral hemodynamics in humans. We aimed to test their feasibility in quantifying CBF, OEF, and CMRO2 in piglets. STUDY TYPE: Prospective. ANIMAL MODEL: Ten neonatal piglets subacutely recovered from global hypoxia-ischemia (N = 2), excitotoxic brain injury (N = 6), or sham procedure (N = 2). FIELD STRENGTH/SEQUENCE: VSASL, TRUPC, and PC MRI acquired at 3.0 T. ASSESSMENT: Regional CBF was measured by VSASL. Global CBF was quantified by both PC and VSASL. TRUPC assessed OEF at the superior sagittal sinus (SSS) and internal cerebral veins (ICVs). CMRO2 was calculated from global CBF and SSS-derived OEF. End-tidal carbon dioxide (EtCO2 ) levels of the piglets were also measured. Brain damage was assessed in tissue sections postmortem by counting damaged neurons. STATISTICAL TESTS: Spearman correlations were performed to evaluate associations among CBF (by PC or VSASL), OEF, CMRO2 , EtCO2 , and the pathological neuron counts. Paired t-test was used to compare OEF at SSS with OEF at ICV. RESULTS: Global CBF was 32.1 ± 14.9 mL/100 g/minute and 30.9 ± 8.3 mL/100 g/minute for PC and VSASL, respectively, showing a significant correlation (r = 0.82, P < 0.05). OEF was 54.9 ± 8.8% at SSS and 46.1 ± 5.6% at ICV, showing a significant difference (P < 0.05). Global CMRO2 was 79.1 ± 26.2 µmol/100 g/minute and 77.2 ± 12.2 µmol/100 g/minute using PC and VSASL-derived CBF, respectively. EtCO2 correlated positively with PC-based CBF (r = 0.81, P < 0.05) but negatively with OEF at SSS (r = -0.84, P < 0.05). Relative CBF of subcortical brain regions and OEF at ICV did not significantly correlate, respectively, with the ratios of degenerating-to-total neurons (P = 0.30, P = 0.10). DATA CONCLUSION: Non-contrast MRI can quantify cerebral hemodynamic parameters in normal and brain-injured neonatal piglets. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY STAGE: 2.

Sulforaphane Protects Piglet Brains from Neonatal Hypoxic-Ischemic Injury.

The striatal, primary sensorimotor cortical, and thalamic neurons are highly vulnerable to hypoxia-ischemia (HI) in term newborns. In a piglet model of HI that exhibits similar selective regional vulnerability, we tested the hypothesis that early treatment with sulforaphane, an activator of the Nrf2 transcription factor, protects vulnerable neurons from HI injury. Anesthetized piglets (aged 3-7 days) were subjected to 45 min of hypoxia and 7 min of airway occlusion. At 15 min after resuscitation, the piglets received intravenous vehicle or sulforaphane. At 4 days of recovery, the density of viable neurons in the putamen of vehicle-treated piglets was 31 ± 34% (±SD) that of sham-operated controls. Treatment with sulforaphane significantly increased viability to 77 ± 31%. In the sensorimotor cortex, neuronal viability was also increased; it was 59 ± 35% in the vehicle-treated and 89 ± 15% in the sulforaphane-treated animals. Treatment with sulforaphane increased the nuclear Nrf2 and γ-glu-tamylcysteine synthetase expression at 6 h of recovery in these regions. We conclude that systemic administration of sulforaphane 15 min after HI can induce the translocation of Nrf2 to the nucleus, increase expression of an enzyme involved in glutathione synthesis, and salvage neurons in the highly vulnerable putamen and sensorimotor cortex in a large-animal model of HI. Therefore, targeting Nrf2 activation soon after recovery from HI is a feasible approach for neuroprotection in the newborn brain.

Neurologic effects of short-term treatment with a soluble epoxide hydrolase inhibitor after cardiac arrest in pediatric swine.

BACKGROUND: Cardiac arrest (CA) is the most common cause of acute neurologic insult in children. Many survivors have significant neurocognitive deficits at 1 year of recovery. Epoxyeicosatrienoic acids (EETs) are multifunctional endogenous lipid signaling molecules that are involved in brain pathobiology and may be therapeutically relevant. However, EETs are rapidly metabolized to less active dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH), limiting their bioavailability. We hypothesized that sEH inhibition would improve outcomes after CA in an infant swine model. Male piglets (3-4 kg, 2 weeks old) underwent hypoxic-asphyxic CA. After resuscitation, they were randomized to intravenous treatment with an sEH inhibitor (TPPU, 1 mg/kg; n = 8) or vehicle (10% poly(ethylene glycol); n = 9) administered at 30 min and 24 h after return of spontaneous circulation. Two sham-operated groups received either TPPU (n = 9) or vehicle (n = 8). Neurons were counted in hematoxylin- and eosin-stained sections from putamen and motor cortex in 4-day survivors. RESULTS: Piglets in the CA + vehicle groups had fewer neurons than sham animals in both putamen and motor cortex. However, the number of neurons after CA did not differ between vehicle- and TPPU-treated groups in either anatomic area. Further, 20% of putamen neurons in the Sham + TPPU group had abnormal morphology, with cell body attrition and nuclear condensation. TPPU treatment also did not reduce neurologic deficits. CONCLUSION: Treatment with an sEH inhibitor at 30 min and 24 h after resuscitation from asphyxic CA does not protect neurons or improve acute neurologic outcomes in piglets.

Mean Diffusivity in Striatum Correlates With Acute Neuronal Death but Not Lesser Neuronal Injury in a Pilot Study of Neonatal Piglets With Encephalopathy.

BACKGROUND: Diffusion MRI is routinely used to evaluate brain injury in neonatal encephalopathy. Although abnormal mean diffusivity (MD) is often attributed to cytotoxic edema, the specific contribution from neuronal pathology is unclear. PURPOSE: To determine whether MD from high-resolution diffusion tensor imaging (DTI) can detect variable degrees of neuronal degeneration and pathology in piglets with brain injury induced by excitotoxicity or global hypoxia-ischemia (HI) with or without overt infarction. STUDY TYPE: Prospective. ANIMAL MODEL: Excitotoxic brain injury was induced in six neonatal piglets by intrastriatal stereotaxic injection of the glutamate receptor agonist quinolinic acid (QA). Three piglets underwent global HI or a sham procedure. Piglets recovered for 20-96 hours before undergoing MRI (n = 9). FIELD STRENGTH/SEQUENCE: 3.0T MRI with DTI, T1 - and T2 -weighted imaging. ASSESSMENT: MD, fractional anisotropy (FA), and qualitative T2 injury were assessed in the putamen and caudate. The cell bodies of normal neurons, degenerating neurons (excitotoxic necrosis, ischemic necrosis, or necrosis-apoptosis cell death continuum), and injured neurons with equivocal degeneration were counted by histopathology. STATISTICAL TESTS: Spearman correlations were used to compare MD and FA to normal, degenerating, and injured neurons. T2 injury and neuron counts were evaluated by descriptive analysis. RESULTS: The QA insult generated titratable levels of neuronal pathology. In QA, HI, and sham piglets, lower MD correlated with higher ratios of degenerating-to-total neurons (P < 0.05), lower ratios of normal-to-total neurons (P < 0.05), and greater numbers of degenerating neurons (P < 0.05). MD did not correlate with abnormal neurons exhibiting nascent injury (P > 0.99). Neuron counts were not related to FA (P > 0.30) or to qualitative injury from T2 -weighted MRI. DATA CONCLUSION: MD is more accurate than FA for detecting neuronal degeneration and loss during acute recovery from neonatal excitotoxic and HI brain injury. MD does not reliably detect nonfulminant, nascent, and potentially reversible neuronal injury. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 2 J. Magn. Reson. Imaging 2020;52:1216-1226.

Administration of a 20-Hydroxyeicosatetraenoic Acid Synthesis Inhibitor Improves Outcome in a Rat Model of Pediatric Traumatic Brain Injury.

The arachidonic acid pathway metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to ischemia/reperfusion brain injury. Inhibition of 20-HETE formation can protect the developing brain from global ischemia. Here, we examined whether treatment with the 20-HETE synthesis inhibitor N-hydroxy-N-4-butyl-2-methylphenylformamidine (HET0016) can protect the immature brain from traumatic brain injury (TBI). Male rats at postnatal day 9-10 underwent controlled cortical impact followed by intraperitoneal injection with vehicle or HET0016 (1 mg/kg, 5 min and 3 h post-injury). HET0016 decreased the lesion volume by over 50% at 3 days of recovery, and this effect persisted at 30 days as the brain matured. HET0016 decreased peri-lesion gene expression of proinflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1ß [IL-1ß]) at 1 day and increased reparative cytokine (IL-4, IL-10) expression at 3 days. It also partially preserved microglial ramified processes, consistent with less activation. HET0016 decreased contralateral hindlimb foot faults and improved outcome on the novel object recognition memory task 30 days after TBI. In cultured BV2 microglia, HET0016 attenuated the lipopolysaccharide-evoked increase in release of TNF-α. Our data show that HET0016 improves acute and long-term histologic and functional outcomes, in association with an attenuated neuroinflammatory response after contusion of an immature rat brain.

Hypoxia-Ischemia and Hypothermia Independently and Interactively Affect Neuronal Pathology in Neonatal Piglets with Short-Term Recovery.

Therapeutic hypothermia is the standard of clinical care for moderate neonatal hypoxic-ischemic encephalopathy. We investigated the independent and interactive effects of hypoxia-ischemia (HI) and temperature on neuronal survival and injury in basal ganglia and cerebral cortex in neonatal piglets. Male piglets were randomized to receive HI injury or sham procedure followed by 29 h of normothermia, sustained hypothermia induced at 2 h, or hypothermia with rewarming during fentanyl-nitrous oxide anesthesia. Viable and injured neurons and apoptotic profiles were counted in the anterior putamen, posterior putamen, and motor cortex at 29 h after HI injury or sham procedure. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) identified genomic DNA fragmentation to confirm cell death. Though hypothermia after HI preserved viable neurons in the anterior and posterior putamen, hypothermia prevented neuronal injury in only the anterior putamen. Hypothermia initiated 2 h after injury did not protect against apoptotic cell death in either the putamen or motor cortex, and rewarming from hypothermia was associated with increased apoptosis in the motor cortex. In non-HI shams, sustained hypothermia during anesthesia was associated with neuronal injury and corresponding viable neuron loss in the anterior putamen and motor cortex. TUNEL confirmed increased neurodegeneration in the putamen of hypothermic shams. Anesthetized, normothermic shams did not show abnormal neuronal cytopathology in the putamen or motor cortex, thereby demonstrating minimal contribution of the anesthetic regimen to neuronal injury during normothermia. We conclude that the efficacy of hypothermic protection after HI is region specific and that hypothermia during anesthesia in the absence of HI may be associated with neuronal injury in the developing brain. Studies examining the potential interactions between hypothermia and anesthesia, as well as with longer durations of hypothermia, are needed.

The Effect of Asphyxia Arrest Duration on a Pediatric End-Tidal CO2-Guided Chest Compression Delivery Model.

OBJECTIVES: To determine the effect of the duration of asphyxial arrest on the survival benefit previously seen with end-tidal CO2-guided chest compression delivery. DESIGN: Preclinical randomized controlled study. SETTING: University animal research laboratory. SUBJECTS: Two-week-old swine. INTERVENTIONS: After either 17 or 23 minutes of asphyxial arrest, animals were randomized to standard cardiopulmonary resuscitation or end-tidal CO2-guided chest compression delivery. Standard cardiopulmonary resuscitation was optimized by marker, monitor, and verbal feedback about compression rate, depth, and release. End-tidal CO2-guided delivery used adjustments to chest compression rate and depth to maximize end-tidal CO2 level without other feedback. Cardiopulmonary resuscitation for both groups proceeded from 10 minutes of basic life support to 10 minutes of advanced life support or return of spontaneous circulation. MEASUREMENTS AND MAIN RESULTS: After 17 minutes of asphyxial arrest, mean end-tidal CO2 during 10 minutes of cardiopulmonary resuscitation was 18 ± 9 torr in the standard group and 33 ± 15 torr in the end-tidal CO2 group (p = 0.004). The rate of return of spontaneous circulation was three of 14 (21%) in the standard group rate and nine of 14 (64%) in the end-tidal CO2 group (p = 0.05). After a 23-minute asphyxial arrest, neither end-tidal CO2 values (20 vs 26) nor return of spontaneous circulation rate (3/14 vs 1/14) differed between the standard and end-tidal CO2-guided groups. CONCLUSIONS: Our previously observed survival benefit of end-tidal CO2-guided chest compression delivery after 20 minutes of asphyxial arrest was confirmed after 17 minutes of asphyxial arrest. The poor survival after 23 minutes of asphyxia shows that the benefit of end-tidal CO2-guided chest compression delivery is limited by severe asphyxia duration.

Abdominal near-infrared spectroscopy in a piglet model of gastrointestinal hypoxia produced by graded hypoxia or superior mesenteric artery ligation.

BackgroundAbdominal near-infrared spectroscopy (aNIRS) may detect gastrointestinal hypoxia before necrotizing enterocolitis develops. We sought to validate aNIRS during splanchnic hypoxia and hypoperfusion in neonatal piglets.MethodsAnesthetized piglets underwent systemic hypoxia or 3 h superior mesenteric artery (SMA) ligation with aNIRS monitoring.ResultsDuring progressive hypoxia, gastrointestinal tissue oxyhemoglobin saturation measured by aNIRS decreased linearly with oxyhemoglobin saturation measured directly in the portal vein. Correlation coefficients were 0.94-0.99 in each of 10 piglets, the average regression slope of 0.73 (95% confidence interval: 0.57, 0.89) differed from one (P<0.004), and the intercept on the aNIRS axis of 9.5% (4.4, 14.6) differed from zero (P<0.0025). Umbilical venous oxyhemoglobin saturation also correlated strongly with the portal vein oxyhemoglobin saturation (r=0.83-0.99), with a slope not different from one. SMA ligation caused ileal blood flow to decrease by ~50%, and produced a sustained decrease in aNIRS oximetry from approximately 60 to 30%.ConclusionaNIRS can detect abrupt and sustained gastrointestinal hypoperfusion associated with arterial occlusion in a neonatal model. The highly linear relationship of portal venous oxyhemoglobin saturation with aNIRS and umbilical vein saturation during graded hypoxia implies that these measures can accurately track tissue oxygenation trends over a wide range in individual subjects.

End-Tidal CO2-Guided Chest Compression Delivery Improves Survival in a Neonatal Asphyxial Cardiac Arrest Model.

OBJECTIVES: To determine whether end-tidal CO2-guided chest compression delivery improves survival over standard cardiopulmonary resuscitation after prolonged asphyxial arrest. DESIGN: Preclinical randomized controlled study. SETTING: University animal research laboratory. SUBJECTS: 1-2-week-old swine. INTERVENTIONS: After undergoing a 20-minute asphyxial arrest, animals received either standard or end-tidal CO2-guided cardiopulmonary resuscitation. In the standard group, chest compression delivery was optimized by video and verbal feedback to maintain the rate, depth, and release within published guidelines. In the end-tidal CO2-guided group, chest compression rate and depth were adjusted to obtain a maximal end-tidal CO2 level without other feedback. Cardiopulmonary resuscitation included 10 minutes of basic life support followed by advanced life support for 10 minutes or until return of spontaneous circulation. MEASUREMENTS AND MAIN RESULTS: Mean end-tidal CO2 at 10 minutes of cardiopulmonary resuscitation was 34 ± 8 torr in the end-tidal CO2 group (n = 14) and 19 ± 9 torr in the standard group (n = 14; p = 0.0001). The return of spontaneous circulation rate was 7 of 14 (50%) in the end-tidal CO2 group and 2 of 14 (14%) in the standard group (p = 0.04). The chest compression rate averaged 143 ± 10/min in the end-tidal CO2 group and 102 ± 2/min in the standard group (p < 0.0001). Neither asphyxia-related hypercarbia nor epinephrine administration confounded the use of end-tidal CO2-guided chest compression delivery. The response of the relaxation arterial pressure and cerebral perfusion pressure to the initial epinephrine administration was greater in the end-tidal CO2 group than in the standard group (p = 0.01 and p = 0.03, respectively). The prevalence of resuscitation-related injuries was similar between groups. CONCLUSIONS: End-tidal CO2-guided chest compression delivery is an effective resuscitation method that improves early survival after prolonged asphyxial arrest in this neonatal piglet model. Optimizing end-tidal CO2 levels during cardiopulmonary resuscitation required that chest compression delivery rate exceed current guidelines. The use of physiologic feedback during cardiopulmonary resuscitation has the potential to provide optimized and individualized resuscitative efforts.

Hypothermia and Rewarming Activate a Macroglial Unfolded Protein Response Independent of Hypoxic-Ischemic Brain Injury in Neonatal Piglets.

Therapeutic hypothermia provides incomplete neuroprotection after hypoxia-ischemia (HI)-induced brain injury in neonates. We previously showed that cortical neuron and white matter apoptosis are promoted by hypothermia and early rewarming in a piglet model of HI. The unfolded protein response (UPR) may be one of the potential mediators of this cell death. Here, neonatal piglets underwent HI or sham surgery followed by 29 h of normothermia, 2 h of normothermia + 27 h of hypothermia or 18 h of hypothermia + rewarming. Piglets recovered for 29 h. Immunohistochemistry for endoplasmic reticulum to nucleus signaling-1 protein (ERN1), a marker of UPR activation, was used to determine the ratios of ERN1+ macroglia and neurons in the motor subcortical white matter and cerebral cortex. The ERN1+ macroglia were immunophenotyped as oligodendrocytes and astrocytes by immunofluorescent colabeling. Temperature (p = 0.046) and HI (p < 0.001) independently affected the ratio of ERN1+ macroglia. In sham piglets, sustained hypothermia (p = 0.011) and rewarming (p = 0.004) increased the ERN1+ macroglia ratio above that in normothermia. HI prior to hypothermia diminished the UPR. Ratios of ERN1+ macroglia correlated with white matter apoptotic profile counts in shams (r = 0.472; p = 0.026), thereby associating UPR activation with white matter apoptosis during hypothermia and rewarming. Accordingly, macroglial cell counts decreased in shams that received sustained hypothermia (p = 0.009) or rewarming (p = 0.007) compared to those in normothermic shams. HI prior to hypothermia neutralized the macroglial cell loss. Neither HI nor temperature affected ERN1+ neuron ratios. In summary, delayed hypothermia and rewarming activate the macroglial UPR, which is associated with white matter apoptosis. HI may decrease the macroglial endoplasmic reticulum stress response after hypothermia and rewarming.

Additive Neuroprotection of a 20-HETE Inhibitor with Delayed Therapeutic Hypothermia after Hypoxia-Ischemia in Neonatal Piglets.

The severity of perinatal hypoxia-ischemia and the delay in initiating therapeutic hypothermia limit the efficacy of hypothermia. After hypoxia-ischemia in neonatal piglets, the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) has been found to contribute to oxidative stress at 3 h of reoxygenation and to eventual neurodegeneration. We tested whether early administration of a 20-HETE synthesis inhibitor after reoxygenation augments neuroprotection with 3-hour delayed hypothermia. In two hypothermic groups, whole body cooling from 38.5 to 34°C was initiated 3 h after hypoxia-ischemia. Rewarming occurred from 20 to 24 h; then anesthesia was discontinued. One hypothermic group received a 20-HETE inhibitor at 5 min after reoxygenation. A sham-operated group and another hypoxia-ischemia group remained normothermic. At 10 days of recovery, resuscitated piglets with delayed hypothermia alone had significantly greater viable neuronal density in the putamen, caudate nucleus, sensorimotor cortex, CA3 hippocampus, and thalamus than did piglets with normothermic recovery, but the values remained less than those in the sham-operated group. In piglets administered the 20-HETE inhibitor before hypothermia, the density of viable neurons in the putamen, cortex and thalamus was significantly greater than in the group with hypothermia alone. Cytochrome P450 4A, which can synthesize 20-HETE, was expressed in piglet neurons in these regions. We conclude that early treatment with a 20-HETE inhibitor enhances the therapeutic benefit of delayed hypothermia in protecting neurons in brain regions known to be particularly vulnerable to hypoxia-ischemia in term newborns.

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.

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.

Noninvasive autoregulation monitoring in a swine model of pediatric cardiac arrest.

BACKGROUND: Cerebrovascular autoregulation after resuscitation has not been well studied in an experimental model of pediatric cardiac arrest. Furthermore, developing noninvasive methods of monitoring autoregulation using near-infrared spectroscopy (NIRS) would be clinically useful in guiding neuroprotective hemodynamic management after pediatric cardiac arrest. We tested the hypotheses that the lower limit of autoregulation (LLA) would shift to a higher arterial blood pressure between 1 and 2 days of recovery after cardiac arrest and that the LLA would be detected by NIRS-derived indices of autoregulation in a swine model of pediatric cardiac arrest. We also tested the hypothesis that autoregulation with hypertension would be impaired after cardiac arrest. METHODS: Data on LLA were obtained from neonatal piglets that had undergone hypoxic-asphyxic cardiac arrest and recovery for 1 day (n = 8) or 2 days (n = 8), or that had undergone sham surgery with 2 days of recovery (n = 8). Autoregulation with hypertension was examined in a separate cohort of piglets that underwent hypoxic-asphyxic cardiac arrest (n = 5) or sham surgery (n = 5) with 2 days of recovery. After the recovery period, piglets were reanesthetized, and autoregulation was monitored by standard laser-Doppler flowmetry and autoregulation indices derived from NIRS (the cerebral oximetry [COx] and hemoglobin volume [HVx] indices). The LLA was determined by decreasing blood pressure through inflation of a balloon catheter in the inferior vena cava. Autoregulation during hypertension was evaluated by inflation of an aortic balloon catheter. RESULTS: The LLAs were similar between sham-operated piglets and piglets that recovered for 1 or 2 days after arrest. The NIRS-derived indices accurately detected the LLA determined by laser-Doppler flowmetry. The area under the curve of the receiver operator characteristic curve for cerebral oximetry index was 0.91 at 1 day and 0.92 at 2 days after arrest. The area under the curve for hemoglobin volume index was 0.92 and 0.89 at the respective time points. During induced hypertension, the static rate of autoregulation, defined as the percentage change in cerebrovascular resistance divided by the percentage change in cerebral perfusion pressure, was not different between postarrest and sham-operated piglets. At 2 days recovery from arrest, piglets exhibited neurobehavioral deficits and histologic neuronal injury. CONCLUSIONS: In a swine model of pediatric hypoxic-asphyxic cardiac arrest with confirmed brain damage, the LLA did not differ 1 and 2 days after resuscitation. The NIRS-derived indices accurately detected the LLA in comparison with laser-Doppler flow measurements at those time points. Autoregulation remained functional during hypertension.

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.

Recovery of baroreflex control of renal sympathetic nerve activity after spinal lesions in the rat.

Spinal cord injury (SCI) has serious long-term consequences on sympathetic cardiovascular regulation. Orthostatic intolerance results from insufficient baroreflex regulation (BR) of sympathetic outflow to maintain proper blood pressure upon postural changes. Autonomic dysreflexia occurs due to insufficient inhibition of spinal sources of sympathetic activity. Both of these conditions result from the inability to control sympathetic activity caudal to SCI. It is well established that limited motor ability recovers after incomplete SCI. Therefore, the goal of this study was to determine whether recovery of BR occurs after chronic, left thoracic spinal cord hemisection at either T(3) or T(8). Baroreflex tests were performed in rats by measuring the reflex response of left (ipsilateral) renal sympathetic nerve activity to decreases and increases in arterial pressure produced by ramped infusions of sodium nitroprusside and phenylephrine, respectively. One week after a T(3) left hemisection, BR function was modestly impaired. However, 8 wk after a T(3) left hemisection, BR function was normal. One week after a T(8) left hemisection, BR function was significantly impaired, and 8 wk after a T(8) left hemisection, BR function was significantly improved. These results indicate that BR of renal sympathetic nerve activity in rats may partially recover after spinal cord hemisections, becoming normal by 8 wk after a T(3) lesion, but not after a T(8) lesion. The nature of the spinal cord and/or brain stem reorganization that mediates this recovery remains to be determined.

Oleuropein Activates Neonatal Neocortical Proteasomes, but Proteasome Gene Targeting by AAV9 Is Variable in a Clinically Relevant Piglet Model of Brain Hypoxia-Ischemia and Hypothermia.

Cerebral hypoxia-ischemia (HI) compromises the proteasome in a clinically relevant neonatal piglet model. Protecting and activating proteasomes could be an adjunct therapy to hypothermia. We investigated whether chymotrypsin-like proteasome activity differs regionally and developmentally in the neonatal brain. We also tested whether neonatal brain proteasomes can be modulated by oleuropein, an experimental pleiotropic neuroprotective drug, or by targeting a proteasome subunit gene using recombinant adeno-associated virus-9 (AAV). During post-HI hypothermia, we treated piglets with oleuropein, used AAV-short hairpin RNA (shRNA) to knock down proteasome activator 28γ (PA28γ), or enforced PA28γ using AAV-PA28γ with green fluorescent protein (GFP). Neonatal neocortex and subcortical white matter had greater proteasome activity than did liver and kidney. Neonatal white matter had higher proteasome activity than did juvenile white matter. Lower arterial pH 1 h after HI correlated with greater subsequent cortical proteasome activity. With increasing brain homogenate protein input into the assay, the initial proteasome activity increased only among shams, whereas HI increased total kinetic proteasome activity. OLE increased the initial neocortical proteasome activity after hypothermia. AAV drove GFP expression, and white matter PA28γ levels correlated with proteasome activity and subunit levels. However, AAV proteasome modulation varied. Thus, neonatal neocortical proteasomes can be pharmacologically activated. HI slows the initial proteasome performance, but then augments ongoing catalytic activity. AAV-mediated genetic manipulation in the piglet brain holds promise, though proteasome gene targeting requires further development.