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LPS Sensitisation Prior to Hypoxia Increases Neuronal Injury and Augments Specific Cytokine Gene Expression Compared to Hypoxia Alone in a Newborn Piglet Model

Presented at the Neonatal Society 2017 Summer Meeting (programme).

Martinello K1, Lingam I1, Advic-Belltheus A1, Meehan C1, Ragab S1, Tachtsidis I1, Hristova M1, Fleiss B2, Peebles D1, Kramer B1, Klein N3, Hagberg H2, Gressens P2, Robertson NJ1

1 Institute for Women’s Health, University College London, London, United Kingdom
2 Centre for the Developing Brain, King’s College London, London, United Kingdom
3 Infectious Diseases and Immunology, University College London, London, United Kingdom

Background: Infection and inflammation contribute to the pathogenesis of neonatal hypoxic ischaemic brain injury, and increase the risk of death and neurodevelopmental impairment for infants with neonatal encephalopathy (1,2). We hypothesise that (i) sensitisation by LPS prior to a global hypoxic injury will worsen brain injury; (ii) a pattern of chemokine and cytokine gene expression will differentiate infection sensitised hypoxia from hypoxa alone.

Methods: 16 Large White male newborn piglets were randomised to i) LPS 2mcg/kg bolus and 1mcg/kg continuous infusion (LPS, n=5); ii) Normal saline bolus and infusion, with hypoxic insult 4 hours after bolus (time 0) (Hypoxia, n=6); and iii) LPS bolus and infusion with hypoxic insult 4 hours after LPS bolus (LPS+Hypoxia, n=5). The hypoxic insult was titrated to BP, EEG and cytochrome c oxidase to ensure similar cerebral insults. Serum samples were taken at baseline, 4h after LPS and at 1, 3, 6, 12, 24 and 48h. RNA was extracted using a mirVANA mRNA isolation kit, converted to cDNA and amplified by PCR. Porcine-specific primers were used and relative mRNA expression (RQ) was evaluated using the comparative CT method. Animals were sacrificed at 48h and regional TUNEL positive counts assessed. This project was funded by the MRC, and conducted according to the UK Home Office Guidelines Animals (Scientific procedures) Act, 1986.

Results: LPS+hypoxia resulted in an increase in mortality from 0% to 60% (p=0.04)(1 died after the hypoxia and 2 died after 24h). Neuronal cell death, as measured by TUNEL count, was greater in the LPS + hypoxia group then the combined total of the LPS alone and hypoxia alone groups (p <0.001, p=0.041), suggesting an exacerbation of brain injury with the combination of LPS and hypoxia. LPS+hypoxia resulted in an up-regulation of genes encoding brain derived neurotrophic factor (RQ 5.4, p=0.04) and microtubule associated protein Tau (RQ 4.1, p=0.03) at 3 hours; this was not seen in any other group. Inflammatory response to LPS, with or without hypoxia, was evidenced by increased expression of CCL2, IL1a, IL8 and IL10. IL1a expression at 3 hours, was significantly greater in the two LPS groups, compared with hypoxia alone piglets (p=0.04). Similarly IL8 was upregulated at 3 and 6 hours in the LPS (RQ 5.7 and 4.6, p=0.01) and LPS+hypoxia (RQ 5.5 and 8.3, p<0.0001) groups, while it was down-regulated in the hypoxia only group at 12 hours (RQ=0.3, p=0.03).

Conclusion: Combined LPS and hypoxia exacerbates neuronal cell death and mortality in the piglet undergoing intensive care and confirms previous rodent studies (3). A specific pattern of inflammatory chemokine gene expression is seen within the first 6h with inflammation (with and without hypoxia) and this pattern differs to hypoxia alone.

Corresponding author:

1. Nelson K, Am J Obstet Gynaecol. 1998;179(2):507-13
2. Tann C, Martinello K. Clin Infec Dis. 2017 In press
3. Eklind et al., Eur J Neurosci 2001;13(6):1101-6

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