Early TLR4 inhibition reduces hippocampal injury at puberty in a rat model of neonatal hypoxic-ischemic brain damage via regulation of neuroimmunity and synaptic plasticity

Neonatal hypoxic-ischemic brain damage (HIBD) survivors present with long-term neurological disorders affecting their quality of life, and there remains a lack of effective treatment. Toll-like receptor 4 (TLR4) is widely distributed in nerve cells and its inhibition has a neuroprotective effect against brain injury. The present study aimed to evaluate the long-term neuroprotective effects of early inhibition of TLR4 during HIBD. Seven-day-old rat pups were subjected to left carotid artery ligation followed by 2 h of hypoxia (8.0% O-2). A single dose of TAK-242 (0.5 mg/kg), a TLR4-specific antagonist, was intraperitoneally injected half an hour prior to hypoxic ischemia (HI). The long-term effects of TAK-242 inhibition on the induced hippocampal injury were investigated by assessing behaviour at P28, and then using a variety of methods to exploring the mechanism, including immunofluorescence, Golgi silver staining, Western blotting and real-time polymerase chain reaction (RT-PCR). TAK-242 treatment significantly reduced the expression levels of TLR4 and its downstream signalling molecules in the ipsilateral lesion of the hippocampus 24 h after HIBD. The Morris water maze (MWM) test demonstrated that TAK-242 treatment reduced the loss of HI-induced learning and memory functions. Immunofluorescence experiments showed that TAK-242 administration attenuated HI-induced loss of neurons, prevented the activation of microglia and astrocytes, and increased the expression of the glutamate receptor subtype, N-methyl D-aspartate 2A (NR2A) in the ipsilateral hippocampus region. Golgi silver staining revealed that TAK-242 prevented an HI-induced decline in spine density in the ipsilateral hippocampus. Western blot and RT-PCR results indicated that the expression of NR2A protein and mRNA in the ipsilateral hippocampi of adolescent rats decreased after neonatal HIBD; early TAK-242 administration may reverse these effects. In conclusion, our findings indicate that early inhibition of TLR4 signalling may improve the long-term prognosis of neonatal HIBD. The mechanisms contributing to this improvement involve reductions in neuronal loss, a decrease in glial cell activation, and an improvement in synaptic plasticity.