K Broad

Brain Cell Death Is Reduced With Cooling by 3.5°C to 5°C but Increased With Cooling by 8.5°C in a Piglet Asphyxia Model

Background and Purpose— In infants with moderate to severe neonatal encephalopathy, whole-body cooling at 33°C to 34°C for 72 hours is standard care with a number needed to treat to prevent a adverse outcome of 6 to 7. The precise brain temperature providing optimal neuroprotection is unknown. Methods— After a quantified global cerebral hypoxic-ischemic insult, 28 piglets aged <24 hours were randomized (each group, n=7) to (1) normothermia (38.5°C throughout) or whole-body cooling 2 to 26 hours after insult to (2) 35°C, (3) 33.5°C, or (4) 30°C. At 48 hours after hypoxia-ischemia, delayed cell death (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling and cleaved caspase 3) and microglial ramification (ionized calcium-binding adapter molecule 1) were evaluated. Results— At 48 hours after hypoxia-ischemia, substantial cerebral injury was found in the normothermia and 30°C hypothermia groups. However, with 35°C and 33.5°C cooling, a clear reduction in delayed cell death and microglial activation was observed in most brain regions (P<0.05), with no differences between 35°C and 33.5°C cooling groups. A protective pattern was observed, with U-shaped temperature dependence in delayed cell death in periventricular white matter, caudate nucleus, putamen, hippocampus, and thalamus. A microglial activation pattern was also seen, with inverted U-shaped temperature dependence in periventricular white matter, caudate nucleus, internal capsule, and hippocampus (all P<0.05). Conclusions— Cooling to 35°C (an absolute drop of 3.5°C as in therapeutic hypothermia protocols) or to 33.5°C provided protection in most brain regions after a cerebral hypoxic-ischemic insult in the newborn piglet. Although the relatively wide therapeutic range of a 3.5°C to 5°C drop in temperature reassured, overcooling (an 8.5°C drop) was clearly detrimental in some brain regions.

Brain mitochondrial oxidative metabolism during and after cerebral hypoxia-ischemia studied by simultaneous phosphorus magnetic-resonance and broadband near-infrared spectroscopy☆

Background Multimodal measurements combining broadband near-infrared spectroscopy (NIRS) and phosphorus magnetic resonance spectroscopy (31P MRS) assessed associations between changes in the oxidation state of cerebral mitochondrial cytochrome-c-oxidase (Δ[oxCCO]) and 31P metabolite peak-area ratios during and after transient cerebral hypoxia–ischemia (HI) in the newborn piglet. Methods Twenty-four piglets (aged < 24 h) underwent transient HI (inspired oxygen fraction 9% and bilateral carotid artery occlusion for ~ 20 min). Whole-brain 31P MRS and NIRS data were acquired every minute. Inorganic phosphate (Pi)/epp, phosphocreatine (PCr)/epp, and total nucleotide triphosphate (NTP)/epp were measured by 31P MRS and were plotted against Δ[oxCCO] during HI and recovery (epp = exchangeable phosphate pool = Pi + PCr + 2γ-NTP + β-NTP). Results During HI Δ[oxCCO], PCr/epp and NTP/epp declined and Pi/epp increased. Significant correlations were seen between 31P ratios and Δ[oxCCO]; during HI a threshold point was identified where the relationship between Δ[oxCCO] and both NTP/epp and Pi/epp changed significantly. Outcome at 48 h related to recovery of Δ[oxCCO] and 31P ratios 1 h post-HI (survived: 1-h NTP/epp 0.22 ± 0.02, Δ[oxCCO] − 0.29 ± 0.50 μM; died: 1-h NTP/epp 0.10 ± 0.04, Δ[oxCCO] − 2.41 ± 1.48 μM). Conclusions Both lowered Δ[oxCCO] and NTP/epp 1 h post-HI indicated mitochondrial impairment. Animals dying before 48 h had slower recovery of both Δ[oxCCO] and 31P ratios by 1 h after HI.

Pharmacokinetics of dexmedetomidine combined with therapeutic hypothermia in a piglet asphyxia model

The highly selective α2‐adrenoreceptor agonist, dexmedetomidine, exerts neuroprotective, analgesic, anti‐inflammatory and sympatholytic properties that may be beneficial for perinatal asphyxia. The optimal safe dose for pre‐clinical newborn neuroprotection studies is unknown.

Inhaled 45–50% argon augments hypothermic brain protection in a piglet model of perinatal asphyxia

Cooling to 33.5 °C in babies with neonatal encephalopathy significantly reduces death and disability, however additional therapies are needed to maximize brain protection. Following hypoxia–ischemia we assessed whether inhaled 45–50% Argon from 2–26 h augmented hypothermia neuroprotection in a neonatal piglet model, using MRS and aEEG, which predict outcome in babies with neonatal encephalopathy, and immunohistochemistry. Following cerebral hypoxia–ischemia, 20 Newborn male Large White piglets < 40 h were randomized to: (i) Cooling (33 °C) from 2–26 h (n = 10); or (ii) Cooling and inhaled 45–50% Argon (Cooling + Argon) from 2–26 h (n = 8). Whole-brain phosphorus-31 and regional proton MRS were acquired at baseline, 24 and 48 h after hypoxia–ischemia. EEG was monitored. At 48 h after hypoxia–ischemia, cell death (TUNEL) was evaluated over 7 brain regions. There were no differences in body weight, duration of hypoxia–ischemia or insult severity; throughout the study there were no differences in heart rate, arterial blood pressure, blood biochemistry and inotrope support. Two piglets in the Cooling + Argon group were excluded. Comparing Cooling + Argon with Cooling there was preservation of whole-brain MRS ATP and PCr/Pi at 48 h after hypoxia–ischemia (p < 0.001 for both) and lower 1H MRS lactate/N acetyl aspartate in white (p = 0.03 and 0.04) but not gray matter at 24 and 48 h. EEG background recovery was faster (p < 0.01) with Cooling + Argon. An overall difference between average cell-death of Cooling versus Cooling + Argon was observed (p < 0.01); estimated cells per mm2 were 23.9 points lower (95% C.I. 7.3–40.5) for the Cooling + Argon versus Cooling. Inhaled 45–50% Argon from 2–26 h augmented hypothermic protection at 48 h after hypoxia–ischemia shown by improved brain energy metabolism on MRS, faster EEG recovery and reduced cell death on TUNEL. Argon may provide a cheap and practical therapy to augment cooling for neonatal encephalopathy.

Immediate remote ischemic postconditioning after hypoxia ischemia in piglets protects cerebral white matter but not grey matter

Remote ischemic postconditioning (RIPostC) is a promising therapeutic intervention whereby brief episodes of ischemia/reperfusion of one organ (limb) mitigate damage in another organ (brain) that has experienced severe hypoxia-ischemia. Our aim was to assess whether RIPostC is protective following cerebral hypoxia-ischemia in a piglet model of neonatal encephalopathy (NE) using magnetic resonance spectroscopy (MRS) biomarkers and immunohistochemistry. After hypoxia-ischemia (HI), 16 Large White female newborn piglets were randomized to: (i) no intervention (n = 8); (ii) RIPostC – with four, 10-min cycles of bilateral lower limb ischemia/reperfusion immediately after HI (n = 8). RIPostC reduced the hypoxic-ischemic-induced increase in white matter proton MRS lactate/N acetyl aspartate (p = 0.005) and increased whole brain phosphorus-31 MRS ATP (p = 0.039) over the 48 h after HI. Cell death was reduced with RIPostC in the periventricular white matter (p = 0.03), internal capsule (p = 0.002) and corpus callosum (p = 0.021); there was reduced microglial activation in corpus callosum (p = 0.001) and more surviving oligodendrocytes in corpus callosum (p = 0.029) and periventricular white matter (p = 0.001). Changes in gene expression were detected in the white matter at 48 h, including KATP channel and endothelin A receptor. Immediate RIPostC is a potentially safe and promising brain protective therapy for babies with NE with protection in white but not grey matter.

Dexmedetomidine combined with therapeutic hypothermia is associated with cardiovascular instability and neurotoxicity in a piglet model of perinatal asphyxia

The selective α2-adrenoreceptor agonist dexmedetomidine has shown neuroprotective, analgesic, anti-inflammatory, and sympatholytic properties that may be beneficial in neonatal encephalopathy (NE). As therapeutic hypothermia is only partially effective, adjunct therapies are needed to optimize outcomes. The aim was to assess whether hypothermia + dexmedetomidine treatment augments neuroprotection compared to routine treatment (hypothermia + fentanyl sedation) in a piglet model of NE using magnetic resonance spectroscopy (MRS) biomarkers, which predict outcomes in babies with NE, and immunohistochemistry. After hypoxia-ischaemia (HI), 20 large White male piglets were randomized to: (i) hypothermia + fentanyl with cooling to 33.5°C from 2 to 26 h, or (ii) hypothermia + dexmedetomidine (a loading dose of 2 μg/kg at 10 min followed by 0.028 μg/kg/h for 48 h). Whole-brain phosphorus-31 and regional proton MRS biomarkers were assessed at baseline, 24, and 48 h after HI. At 48 h, cell death was evaluated over 7 brain regions by means of transferase-mediated d-UTP nick end labeling (TUNEL). Dexmedetomidine plasma levels were mainly within the target sedative range of 1 μg/L. In the hypothermia + dexmedetomidine group, there were 6 cardiac arrests (3 fatal) versus 2 (non-fatal) in the hypothermia + fentanyl group. The hypothermia + dexmedetomidine group required more saline (p = 0.005) to maintain blood pressure. Thalamic and white-matter lactate/N-acetylaspartate did not differ between groups (p = 0.66 and p = 0.21, respectively); the whole-brain nucleotide triphosphate/exchangeable phosphate pool was similar (p = 0.73) over 48 h. Cell death (TUNEL-positive cells/mm2) was higher in the hypothermia + dexmedetomidine group than in the hypothermia + fentanyl group (mean 5.1 vs. 2.3, difference 2.8 [95% CI 0.6-4.9], p = 0.036). Hypothermia + dexmedetomidine treatment was associated with adverse cardiovascular events, even within the recommended clinical sedative plasma level; these may have been exacerbated by an interaction with either isoflurane or low body temperature. Hypothermia + dexmedetomidine treatment was neurotoxic following HI in our piglet NE model, suggesting that caution is vital if dexmedetomidine is combined with cooling following NE.

Ultramicronized palmitoylethanolamide rescues learning and memory impairments in a triple transgenic mouse model of Alzheimer's disease by exerting anti-inflammatory and neuroprotective effects

In an aging society, Alzheimer’s disease (AD) exerts an increasingly serious health and economic burden. Current treatments provide inadequate symptomatic relief as several distinct pathological processes are thought to underlie the decline of cognitive and neural function seen in AD. This suggests that the efficacy of treatment requires a multitargeted approach. In this context, palmitoylethanolamide (PEA) provides a novel potential adjunct therapy that can be incorporated into a multitargeted treatment strategy. We used young (6-month-old) and adult (12-month-old) 3×Tg-AD mice that received ultramicronized PEA (um-PEA) for 3 months via a subcutaneous delivery system. Mice were tested with a range of cognitive and noncognitive tasks, scanned with magnetic resonance imaging/magnetic resonance spectroscopy (MRI/MRS), and neurochemical release was assessed by microdialysis. Potential neuropathological mechanisms were assessed postmortem by western blot, reverse transcription–polymerase chain reaction (RT-PCR), and immunofluorescence. Our data demonstrate that um-PEA improves learning and memory, and ameliorates both the depressive and anhedonia-like phenotype of 3×Tg-AD mice. Moreover, it reduces Aβ formation, the phosphorylation of tau proteins, and promotes neuronal survival in the CA1 subregion of the hippocampus. Finally, um-PEA normalizes astrocytic function, rebalances glutamatergic transmission, and restrains neuroinflammation. The efficacy of um-PEA is particularly potent in younger mice, suggesting its potential as an early treatment. These data demonstrate that um-PEA is a novel and effective promising treatment for AD with the potential to be integrated into a multitargeted treatment strategy in combination with other drugs. Um-PEA is already registered for human use. This, in combination with our data, suggests the potential to rapidly proceed to clinical use.

Isoflurane Exposure Induces Cell Death, Microglial Activation and Modifies the Expression of Genes Supporting Neurodevelopment and Cognitive Function in the Male Newborn Piglet Brain.

Exposure of the brain to general anesthesia during early infancy may adversely affect its neural and cognitive development. The mechanisms mediating this are complex, incompletely understood and may be sexually dimorphic, but include developmentally inappropriate apoptosis, inflammation and a disruption to cognitively salient gene expression. We investigated the effects of a 6h isoflurane exposure on cell death, microglial activation and gene expression in the male neonatal piglet brain. Piglets (n = 6) were randomised to: (i) naive controls or (ii) 6h isoflurane. Cell death (TUNEL and caspase-3) and microglial activation were recorded in 7 brain regions. Changes in gene expression (microarray and qPCR) were assessed in the cingulate cortex. Electroencephalography (EEG) was recorded throughout. Isoflurane anesthesia induced significant increases in cell death in the cingulate and insular cortices, caudate nucleus, thalamus, putamen, internal capsule, periventricular white matter and hippocampus. Dying cells included both neurons and oligodendrocytes. Significantly, microglial activation was observed in the insula, pyriform, hippocampus, internal capsule, caudate and thalamus. Isoflurane induced significant disruption to the expression of 79 gene transcripts, of these 26 are important for the control of transcription and 23 are important for the mediation of neural plasticity, memory formation and recall. Our observations confirm that isoflurane increases apoptosis and inflammatory responses in the neonatal piglet brain but also suggests novel additional mechanisms by which isoflurane may induce adverse neural and cognitive development by disrupting the expression of genes mediating activity dependent development of neural circuits, the predictive adaptive responses of the brain, memory formation and recall.

Modelling blood flow and metabolism in the piglet brain during hypoxia-ischaemia: simulating pH changes.

We describe the extension of a computational model of blood flow and metabolism in the piglet brain to investigate changes in neonatal intracellular brain pH during hypoxia-ischemia (HI). The model is able to simulate near-infrared spectroscopy (NIRS) and magnetic resonance spectroscopy (MRS) measurements obtained from HI experiments conducted in piglets. We adopt a method of using 31P-MRS data to estimate of intracellular pH and compare measured pH and oxygenation with their modelled counterparts. We show that both NIRS and MRS measurements are predicted well in the new version of the model.

Surgery increases cell death and induces changes in gene expression compared with anesthesia alone in the developing piglet brain

In a range of animal species, exposure of the brain to general anaesthesia without surgery during early infancy may adversely affect its neural and cognitive development. The mechanisms mediating this are complex but include an increase in brain cell death. In humans, attempts to link adverse cognitive development to infantile anaesthesia exposure have yielded ambiguous results. One caveat that may influence the interpretation of human studies is that infants are not exposed to general anaesthesia without surgery, raising the possibility that surgery itself, may contribute to adverse cognitive development. Using piglets, we investigated whether a minor surgical procedure increases cell death and disrupts neuro-developmental and cognitively salient gene transcription in the neonatal brain. We randomly assigned neonatal male piglets to a group who received 6h of 2% isoflurane anaesthesia or a group who received an identical anaesthesia plus 15 mins of surgery designed to replicate an inguinal hernia repair. Compared to anesthesia alone, surgery-induced significant increases in cell death in eight areas of the brain. Using RNAseq data derived from all 12 piglets per group we also identified significant changes in the expression of 181 gene transcripts induced by surgery in the cingulate cortex, pathway analysis of these changes suggests that surgery influences the thrombin, aldosterone, axonal guidance, B cell, ERK-5, eNOS and GABAA signalling pathways. This suggests a number of novel mechanisms by which surgery may influence neural and cognitive development independently or synergistically with the effects of anaesthesia.