Hypothermic Neuronal Rescue from Infection-Sensitised Hypoxic-Ischaemic Brain Injury Is Pathogen Dependent

Abstract

Perinatal infection increases the vulnerability of the neonatal brain to hypoxic-ischaemic (HI) injury. Hypothermia treatment (HT) does not provide neuroprotection after pre-insult inflammatory sensitisation by lipopolysaccharide (LPS), a gram-negative bacterial wall constituent. However, early-onset sepsis in term babies is caused by gram-positive species in more than 90% of cases, and neuro-inflammatory responses triggered through the gram-negative route (Toll-like receptor 4, TLR-4) are different from those induced through the gram-positive route via TLR-2. Whether gram-positive septicaemia sensitises the neonatal brain to hypoxia and inhibits the neuroprotective effect of HT is unknown. Seven-day-old Wistar rats (n = 178) were subjected to intraperitoneal injections of PAM3CSK4 (1 mg/kg, a synthetic TLR-2 agonist) or vehicle (0.9% NaCl). After an 8-h delay, the left carotid artery was ligated followed by 50 min of hypoxia (8% O2) at a rectal temperature of 36°C. Pups received a 5-h treatment of normothermia (NT, 37°C) or HT (32°C) immediately after the insult. Brains were harvested after 7 days' survival for hemispheric and hippocampal area loss analyses and immunolabelling of microglia (Iba1) and hippocampal neurons (NeuN). Normothermic PAM3CSK4-injected animals showed significantly more brain injury than vehicle animals (p = 0.014). Compared to NT, HT significantly reduced injury in the PAM3CSK4-injected animals, with reduced area loss (p < 0.001), reduced microglial activation (p = 0.006), and increased neuronal rescue in the CA1 region (p < 0.001). Experimental induction of a sepsis-like condition through the gram-positive pathway sensitises the brain to HI injury. HT was highly neuroprotective after the PAM3CSK4-triggered injury, suggesting HT may be neuroprotective in the presence of a gram-positive infection. These results are in strong contrast to LPS studies where HT is not neuroprotective.

Keywords: Asphyxia; Gram-positive pathogens; Hypothermia therapy; Infection; Inflammation; Lipopolysaccharide; Neuroprotection; PAM3CSK4; Perinatal brain injury.