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.

Hypothermia Is Neuroprotective after Severe Hypoxic-Ischaemic Brain Injury in Neonatal Rats Pre-Exposed to PAM₃CSK₄

“…The dose of PAM was based on previous publications on neonatal rodents [13, 18, 19], in combination with our own dose-response experiments where we compared post-insult hemispheric area loss according to our standard protocol [16]. We titrated the dose in combination with 50 min of 8% hypoxia in order to produce what we have previously defined as a moderate degree of brain injury (around 40% area loss of the affected hemisphere), as we have done previously in the LPS-exposed model [15].…”

“…All surgical procedures were performed as previously described [16], but followed by a 90-min instead of a 50-min hypoxic insult. Briefly, at the start of each experiment, animals were injected with PAM or Veh according to randomisation.…”

“…Immunohistochemical staining was performed as described in previous publications using the same material and antibodies [16]. Briefly, slides were prepared from paraffin-embedded sections.…”

“…However, as opposed to in LPS pre-exposed HI brain injury, HT is still highly neuroprotective in PAM pre-exposed HI injury (previous data combined in Fig. 1) [16]. …”

“…HT provided significant neuroprotection after 1 week’s survival in PAM-exposed pups ( p < 0.0002) but not in LPS-exposed pups. Modified and combined from Osredkar et al [15] and Falck et al [16]. …”