Acute LPS sensitization and continuous infusion exacerbates hypoxic brain injury in a piglet model of neonatal encephalopathy

Abstract

Co-existing infection/inflammation and birth asphyxia potentiate the risk of developing neonatal encephalopathy (NE) and adverse outcome. In a newborn piglet model we assessed the effect of E. coli lipopolysaccharide (LPS) infusion started 4 h prior to and continued for 48 h after hypoxia on brain cell death and systemic haematological changes compared to LPS and hypoxia alone. LPS sensitized hypoxia resulted in an increase in mortality and in brain cell death (TUNEL positive cells) throughout the whole brain, and in the internal capsule, periventricular white matter and sensorimotor cortex. LPS alone did not increase brain cell death at 48 h, despite evidence of neuroinflammation, including the greatest increases in microglial proliferation, reactive astrocytosis and cleavage of caspase-3. LPS exposure caused splenic hypertrophy and platelet count suppression. The combination of LPS and hypoxia resulted in the highest and most sustained systemic white cell count increase. These findings highlight the significant contribution of acute inflammation sensitization prior to an asphyxial insult on NE illness severity.

High-Dose Melatonin and Ethanol Excipient Combined with Therapeutic Hypothermia in a Newborn Piglet Asphyxia Model

“…Increased CC3 expression, not linked the cell death, has been observed in the contralateral hemisphere in a unilateral hypoperfusion P7-rat model 49 . We have previously observed discrepancies between TUNEL-positive cell death and CC3 in our piglet model 32 . The use of male piglets may partly explain these data; cell death is dimorphic, and in males, apoptosis occurs via caspase-independent pathways 50,51 .…”

“…For each animal, 2 sections (bregma 00 and −2.0) were stained and 8 brain regions were examined: cingulate cortex, sensorimotor cortex, hippocampus, internal capsule, periventricular white matter, caudate, putamen and thalamus. Histological analysis was performed by an investigator blinded to treatment allocation 32 .…”

Proton Magnetic Resonance Spectroscopy Lactate/N-Acetylaspartate Within 48 h Predicts Cell Death Following Varied Neuroprotective Interventions in a Piglet Model of Hypoxia–Ischemia With and Without Inflammation-Sensitization

“…Piglets were anesthetized and surgically prepared as described previously (11,13,14,16,17). In brief, all piglets were sedated with intramuscular midazolam and anesthetized with inhaled 3-4% v/v isoflurane.…”

“…Piglets were randomized to: (i) vehicle infusion; (ii) magnesium bolus 1 h post insult followed by continuous infusion over 48 h; or (iii) melatonin infused over 2 h at 1 h and 25 h after insult. Study 3 (14): Piglets were randomized to: (i) control (saline bolus and infusion), (ii) lipopolysaccharide (LPS) (bolus followed by a continuous infusion); (iii) hypoxia; or (iv) LPS and hypoxia 4 h after bolus. Study 4: All piglets were subjected to LPS bolus 4 h prior to HI and continuous infusion over 52 h. Animals were then randomized to either normothermia or 12 h HT from 1 to 13 h after insult.…”

“…Abbreviations: 3T, 3-Tesla; ADC, apparent diffusion coefficient; ANLS, astrocyte neuron lactate shuttle; AUC, area under the curve; BGT, basal ganglia, thalamus; CC3, cleaved caspase 3; Cho, choline; Cr, creatine; DGM, Deep gray matter; DWI, diffusion-weighted imaging; FA, fractional anisotropy; HI, hypoxia-ischemia; HT, therapeutic hypothermia; Iba1, ionized calcium-binding adapter molecule 1; Lac/NAA, lactate/N-acetylaspartate; Lac, lactate; LPS, lipopolysaccharide; MD, mean diffusivity; MRS, magnetic resonance spectroscopy; NAA, N-acetylaspartate; NAAG, N-acetylaspartylglutamate; NAD, nicotinamide adenine dinucleotide; NE, neonatal encephalopathy; RCT, randomized controlled trial; ROI, region of Over the last two decades, we have used BGT and white matter (WM) Lac/NAA as one of our primary outcome markers in neuroprotection studies of adjunct therapies with HT in our piglet model (11)(12)(13)(14)(15). The piglet model allows for regional assessment of brain immunohistochemistry at 48 h with analyses including quantification of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, assessment of neuroinflammation [ionized calcium-binding adapter molecule 1 (Iba1) ramification index], and quantification of cleaved caspase 3 (CC3), a marker of caspase-dependent apoptosis.…”