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.

Use of an end-tidal carbon dioxide-guided algorithm during cardiopulmonary resuscitation improves short-term survival in paediatric swine.

AIM: To evaluate an algorithm that uses an end-tidal carbon dioxide (ETCO2) target of ≥ 30 torr to guide specific changes in chest compression rate and epinephrine administration during cardiopulmonary resuscitation (CPR) in paediatric swine. METHODS: Swine underwent asphyxial cardiac arrest followed by resuscitation with either standard or ETCO2-guided algorithm CPR. The standard group received chest compressions at a rate of 100/min and epinephrine every 4 min during advanced life support consistent with the American Heart Association paediatric resuscitation guidelines. In the ETCO2-guided algorithm group, chest compression rate was increased by 10 compressions/min for every minute that the ETCO2 was < 30 torr, and the epinephrine administration interval was decreased to every 2 min if the ETCO2 remained < 30 torr. Short-term survival and physiologic data during active resuscitation were compared. RESULTS: Short-term survival was significantly greater in the ETCO2-guided algorithm CPR group than in the standard CPR group (16/28 [57.1%] versus 4/28 [14.3%]; p = 0.002). Additionally, the algorithm group had higher predicted mean ETCO2, chest compression rate, diastolic and mean arterial pressure, and myocardial perfusion pressure throughout resuscitation. Swine in the algorithm group also exhibited significantly greater improvement in diastolic and mean arterial pressure and cerebral perfusion pressure after the first dose of epinephrine than did those in the standard group. Incidence of resuscitation-related injuries was similar in the two groups. CONCLUSIONS: Use of a resuscitation algorithm with stepwise guidance for changes in the chest compression rate and epinephrine administration interval based on a goal ETCO2 level improved survival and intra-arrest hemodynamics in this porcine cardiac arrest model.

Combining Hypothermia and Oleuropein Subacutely Protects Subcortical White Matter in a Swine Model of Neonatal Hypoxic-Ischemic Encephalopathy.

Neonatal hypoxia-ischemia (HI) causes white matter injury that is not fully prevented by therapeutic hypothermia. Adjuvant treatments are needed. We compared myelination in different piglet white matter regions. We then tested whether oleuropein (OLE) improves neuroprotection in 2- to 4-day-old piglets randomized to undergo HI or sham procedure and OLE or vehicle administration beginning at 15 minutes. All groups received overnight hypothermia and rewarming. Injury in the subcortical white matter, corpus callosum, internal capsule, putamen, and motor cortex gray matter was assessed 1 day later. At baseline, piglets had greater subcortical myelination than in corpus callosum. Hypothermic HI piglets had scant injury in putamen and cerebral cortex. However, hypothermia alone did not prevent the loss of subcortical myelinating oligodendrocytes or the reduction in subcortical myelin density after HI. Combining OLE with hypothermia improved post-HI subcortical white matter protection by preserving myelinating oligodendrocytes, myelin density, and oligodendrocyte markers. Corpus callosum and internal capsule showed little HI injury after hypothermia, and OLE accordingly had minimal effect. OLE did not affect putamen or motor cortex neuron counts. Thus, OLE combined with hypothermia protected subcortical white matter after HI. As an adjuvant to hypothermia, OLE may subacutely improve regional white matter protection after HI.

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.

Association of diastolic blood pressure with survival during paediatric cardiopulmonary resuscitation.

AIM: To examine the relationship between survival and diastolic blood pressure (DBP) throughout resuscitation from paediatric asphyxial cardiac arrest. METHODS: Retrospective, secondary analysis of 200 swine resuscitations. Swine underwent asphyxial cardiac arrest and were resuscitated with predefined periods of basic and advanced life support (BLS and ALS, respectively). DBP was recorded every 30 s. Survival was defined as 20-min sustained return of spontaneous circulation (ROSC). RESULTS: During BLS, DBP peaked between 1-3 min and was greater in survivors (20.0 [11.3, 33.3] mmHg) than in non-survivors (5.0 [1.0, 10.0] mmHg; p < 0.001). After this transient increase, the DBP in survivors progressively decreased but remained greater than that of non-survivors after 10 min of resuscitation (9.0 [6.0, 13.8] versus 3.0 [1.0, 6.8] mmHg; p < 0.001). During ALS, the magnitude of DBP change after the first adrenaline (epinephrine) administration was greater in survivors (22.0 [16.5, 36.5] mmHg) than in non-survivors (6.0 [2.0, 11.0] mmHg; p < 0.001). Survival rate was greater when DBP improved by ≥26 mmHg after the first dose of adrenaline (20/21; 95%) than when DBP improved by ≤10 mmHg (1/99; 1%). The magnitude of DBP change after the first adrenaline administration correlated with the timetoROSC (r = -0.54; p < 0.001). CONCLUSIONS: Survival after asphyxial cardiac arrest is associated with a higher DBP throughout resuscitation, but the difference between survivors and non-survivors was reduced after prolonged BLS. During ALS, response to adrenaline administration correlates with survival and time to ROSC. If confirmed clinically, these findings may be useful for titrating adrenaline during resuscitation and prognosticating likelihood of ROSC. Institutional Protocol Numbers: SW14M223 and SW17M101.

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.

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.

Spatial T-maze identifies cognitive deficits in piglets 1 month after hypoxia-ischemia in a model of hippocampal pyramidal neuron loss and interneuron attrition.

Neonatal brain injury from hypoxia-ischemia (HI) causes major morbidity. Piglet HI is an established method for testing neuroprotective treatments in large, gyrencephalic brain. Though many neurobehavior tests exist for rodents, such tests and their associations with neuropathologic injury remain underdeveloped and underutilized in large, neonatal HI animal models. We examined whether spatial T-maze and inclined beam tests distinguish cognitive and motor differences between HI and sham piglets and correlate with neuropathologic injury. Neonatal piglets were randomized to whole-body HI or sham procedure, and they began T-maze and inclined beam testing 17 days later. HI piglets had more incorrect T-maze turns than did shams. Beam walking time did not differ between groups. Neuropathologic evaluations at 33 days validated the injury with putamen neuron loss after HI to below that of sham procedure. HI decreased the numbers of CA3 pyramidal neurons but not CA1 pyramidal neurons or dentate gyrus granule neurons. Though the number of hippocampal parvalbumin-positive interneurons did not differ between groups, HI reduced the number of CA1 interneuron dendrites. Piglets with more incorrect turns had greater CA3 neuron loss, and piglets that took longer in the maze had fewer CA3 interneurons. The number of putamen neurons was unrelated to T-maze or beam performance. We conclude that neonatal HI causes hippocampal CA3 neuron loss, CA1 interneuron dendritic attrition, and putamen neuron loss at 1-month recovery. The spatial T-maze identifies learning and memory deficits that are related to loss of CA3 pyramidal neurons and fewer parvalbumin-positive interneurons independent of putamen injury.

Proteasome Biology Is Compromised in White Matter After Asphyxic Cardiac Arrest in Neonatal Piglets.

Background Neurological deficits in hypoxic-ischemic encephalopathy, even with therapeutic hypothermia, are partially attributed to white matter injury. We theorized that proteasome insufficiency contributes to white matter injury. Methods and Results Neonatal piglets received hypoxia-ischemia ( HI ) or sham procedure with normothermia, hypothermia, or hypothermia+rewarming. Some received a proteasome activator drug (oleuropein) or white matter-targeted, virus-mediated proteasome knockdown. We measured myelin oligodendrocyte glycoprotein, proteasome subunit 20S (P20S), proteasome activity, and carbonylated and ubiquitinated protein levels in white matter and cerebral cortex. HI reduced myelin oligodendrocyte glycoprotein levels regardless of temperature, and myelin oligodendrocyte glycoprotein loss was associated with increased ubiquitinated and carbonylated protein levels. Ubiquitinated and carbonyl-damaged proteins increased in white matter 29 hours after HI during hypothermia to exceed levels at 6 to 20 hours. In cortex, ubiquitinated proteins decreased. Ubiquitinated and carbonylated protein accumulation coincided with lower P20S levels in white matter; P20S levels also decreased in cerebral cortex. However, proteasome activity in white matter lagged behind that in cortex 29 hours after HI during hypothermia. Systemic oleuropein enhanced white matter P20S and protected the myelin, whereas proteasome knockdown exacerbated myelin oligodendrocyte glycoprotein loss and ubiquitinated protein accumulation after HI . At the cellular level, temperature and HI interactively affected macroglial P20S enrichment in subcortical white matter. Rewarming alone increased macroglial P20S immunoreactivity, but this increase was blocked by HI . Conclusions Oxidized and ubiquitinated proteins accumulate with HI -induced white matter injury. Proteasome insufficiency may drive this injury. Hypothermia did not prevent myelin damage, protect the proteasome, or preserve oxidized and ubiquitinated protein clearance after HI .

Pilot Study to Compare the Use of End-Tidal Carbon Dioxide-Guided and Diastolic Blood Pressure-Guided Chest Compression Delivery in a Swine Model of Neonatal Asphyxial Cardiac Arrest.

Background The American Heart Association recommends use of physiologic feedback when available to optimize chest compression delivery. We compared hemodynamic parameters during cardiopulmonary resuscitation in which either end-tidal carbon dioxide ( ETCO 2) or diastolic blood pressure ( DBP ) levels were used to guide chest compression delivery after asphyxial cardiac arrest. Methods and Results One- to 2-week-old swine underwent a 17-minute asphyxial-fibrillatory cardiac arrest followed by alternating 2-minute periods of ETCO 2-guided and DBP -guided chest compressions during 10 minutes of basic life support and 10 minutes of advanced life support. Ten animals underwent resuscitation. We found significant changes to ETCO 2 and DBP levels within 30 s of switching chest compression delivery methods. The overall mean ETCO 2 level was greater during ETCO 2-guided cardiopulmonary resuscitation (26.4±5.6 versus 22.5±5.2 mm Hg; P=0.003), whereas the overall mean DBP was greater during DBP -guided cardiopulmonary resuscitation (13.9±2.3 versus 9.4±2.6 mm Hg; P=0.003). ETCO 2-guided chest compressions resulted in a faster compression rate (149±3 versus 120±5 compressions/min; P=0.0001) and a higher intracranial pressure (21.7±2.3 versus 16.0±1.1 mm Hg; P=0.002). DBP -guided chest compressions were associated with a higher myocardial perfusion pressure (6.0±2.8 versus 2.4±3.2; P=0.02) and cerebral perfusion pressure (9.0±3.0 versus 5.5±4.3; P=0.047). Conclusions Using the ETCO 2 or DBP level to optimize chest compression delivery results in physiologic changes that are method-specific and occur within 30 s. Additional studies are needed to develop protocols for the use of these potentially conflicting physiologic targets to improve outcomes of prolonged cardiopulmonary resuscitation.

d-Amino Acid Levels in Perfused Mouse Brain Tissue and Blood: A Comparative Study.

The l-enantiomer is the predominant type of amino acid in all living systems. However, d-amino acids, once thought to be "unnatural", have been found to be indigenous even in mammalian systems and increasingly appear to be functioning in essential biological and neurological roles. Both d- and l-amino acid levels in the hippocampus, cortex, and blood samples from NIH Swiss mice are reported. Perfused brain tissues were analyzed for the first time, thereby eliminating artifacts due to endogenous blood, and decreased the mouse-to-mouse variability in amino acid levels. Total amino acid levels (l- plus d-enantiomers) in brain tissue are up to 10 times higher than in blood. However, all measured d-amino acid levels in brain tissue are typically ∼10 to 2000 times higher than blood levels. There was a 13% reduction in almost all measured d-amino acid levels in the cortex compared to those in the hippocampus. There is an approximate inverse relationship between the prevalence of an amino acid and the percentage of its d-enantiomeric form. Interestingly, glutamic acid, unlike all other amino acids, had no quantifiable level of its d-antipode. The bioneurological reason for the unique and conspicuous absence/removal of this d-amino acid is yet unknown. However, results suggest that d-glutamate metabolism is likely a unidirectional process and not a cycle, as per the l-glutamate/glutamine cycle. The results suggest that there might be unreported d-amino acid racemases in mammalian brains. The regulation and function of specific other d-amino acids are discussed.

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.