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.

Figure 1

TUNEL histology. There was an overall increase in the estimated mean TUNEL+ cells per mm² (pooled across region and R0/R1 levels) in the LPS + Hypoxia group versus all other groups (a). Representative sections are shown at x40 magnification from the internal capsule (b). A TUNEL+ cell is arrowed. On regional assessment, there was an increase in TUNEL+ cells for LPS + Hypoxia in the IC and PvWM versus Naïve, Sham and LPS, and in the sCTX versus Naïve and LPS (c). Cingulate cortex = cCTX; Sensorimotor cortex = sCTX; Hippocampus = HIP; Periventricular white matter = PvWM; Internal capsule = IC; Caudate = CAUD; Putamen = PTMN; Thalamus = THAL. Error bars represent standard error. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 2

Estimated mean whole brain (left) and regional (right) cleaved caspase-3 (a), IBA1 cell count (b), IBA1 ramification index (c) and GFAP luminosity (d). CC3 positive cells markedly increased with LPS exposure. There was no effect of hypoxia in isolation on CC3. In combination, the increase in CC3 with LPS was attenuated by hypoxia exposure. Microglia number (IBA1 count) was increased by LPS exposure, whilst microglial activation (IBA1 ramification index) was increased by hypoxia exposure. Astrogliosis was increased from Naïve for all groups, maximal for LPS. Cingulate cortex = cCTX; Sensorimotor cortex = sCTX; Hippocampus = HIP; Periventricular white matter = PvWM; Internal capsule = IC; Caudate = CAUD; Putamen = PTMN; Thalamus = THAL. Error bars represent standard error. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 3

Representative IBA1 stained microglia in the cingulate cortex at x40 magnification, for Naïve (a), Sham (b), LPS (c), Hypoxia (d) and LPS + Hypoxia (e). In non-disease states, microglia have small cell bodies and multiple fine branches. LPS exposure subjectively increased branch thickness, while hypoxia induced amoeboid change.

Figure 4

Mean aEEG score from baseline (BL) till 48 h, divided into 6 h time epochs (a), using pattern classification scoring system (b), where 0 = flat trace, 1 = low voltage, 2 = burst suppression, 3 = discontinuous normal voltage, and 4 = continuous normal voltage. After insult, Hypoxia group had lower scores than Sham at all time points (p ≤ 0.025), and than LPS until 36 h (p ≤ 0.021). LPS + Hypoxia had lower scores than Sham at all time points, except 25–30 h (p ≤ 0.038); and than LPS from 0–12 h, and again at 43–48 h (p ≤ 0.033). There was no difference between Hypoxia and LPS + Hypoxia. Phenobarbitone was given to one Hypoxia piglet at 6 h post insult (20 mg/kg), and one LPS + Hypoxia piglet at 22 h (20 mg/kg) and 39 h (10 mg/kg) post insult. Error bars represent standard error. *p < 0.05 vs. Sham (*colour denotes group), ^†p < 0.05 LPS vs. Hypoxia, ^‡p < 0.05 LPS vs. LPS + Hypoxia. Yellow arrows along the x-axis represent approximate time of death of the 3 LPS + Hypoxia subjects who died prematurely.

Figure 5

Change in haematology parameters throughout experimentation. WCC and platelet count analysis included between group comparisons at each time point. Error bars represent standard error. BL = baseline; β p < 0.05 change from BL (β colour denotes group), *p < 0.05 vs. Sham at same time point (*colour denotes group), ^†p < 0.05 LPS vs. Hypoxia, ^‡p < 0.05 LPS vs. LPS + Hypoxia, ^#p < 0.05 Hypoxia vs. LPS + Hypoxia. Yellow arrows along the x-axis represent approximate time of death of the 3 LPS + Hypoxia subjects who died prematurely.

Figure 6

Study time-line. Following initial weight and wellbeing assessment, 7 piglets were randomly assigned to immediate euthanasia (Naïve). Following baseline recordings and tracheostomy, remaining piglets were randomized to receive (i) Saline bolus and infusion alone (Sham, n = 3), (ii) LPS bolus and infusion alone (LPS, n = 5), (iv) hypoxia 4 h after saline bolus (Hypoxia, n = 6) and (v) hypoxia 4 h after LPS bolus (LPS + Hypoxia, n = 5). Blood samples were collected at baseline, 4 h after bolus, end of hypoxia (time 0), and at 3, 12, 24 and 48 h after time 0. Piglets were maintained under meticulous intensive care for 48 h following hypoxia, prior to euthanasia and histological specimen collection. EEG was acquired continuously throughout.