Abstract 3348: A Novel Role for TLR-3 in Protecting the Immature Brain from Ischemic Injury

Abstract

Background: We have shown that giving a low dose of lipopolysaccharide (LPS) to P7 rat pups will result in 90% reduction in ischemia-induced brain damage. This dramatic LPS neuroprotection was observed in P7rat pups which correspond in terms of brain development to human term newborn but not in P3 or P5 pups which correspond to human premature infant. LPS is a known Toll-like receptor 4 (TLR-4) ligand. We hypothesized that TLRs other than TLR-4 may mediate preconditioning against cerebral ischemic injury in the developing brain Methods: Brains from P5 and P7 rats were removed, fixed in 10% formalin, embedded in paraffin and cut in 5μ m coronal sections. TLR-3 and TLR-4 expression was assessed by immunohistochemistry. In subsequent experiment, P5 Rat pups were randomly assigned to TLR-3 specific agonist, poly I:C, treated group (20μ g; IP) or saline treated group. Forty eight hours after the injections, hypoxic-ischemic injury was induced by using the Rice-Vannucci model. Unilateral internal carotid artery ligation in rat pups followed by exposure to 8% hypoxia for approximately 65min will cause a reproducible unilateral infarct ipsilateral to the ligated artery involving caudate, putamen, hippocampus and cortex. Body temperature will be maintained at 37-37.5 0 C during hypoxia using an incubator. Brains were removed 1 week after HI injury, fixed, embedded in paraffin and cut in 5μ m coronal sections. Brain sections were then stained with Hematoxylin and Eosin and infarct volumes were compared between the two groups using Nikon NIS-Element Basic Research Image analysis software system Results: TLR-4 was highly expressed in brains of P7 but not in P5 rat pups. The number of TLR-4 positive cells was significantly lower in P5 rat brains compared to P7 pups ( p < 0.05). TLR-3 was predominately expressed in P5 ( p < 0.05). There was a significant reduction in infarct volume ( p = 0.01) in poly I:C P5 treated pups (6.6mm 3 + SEM 3) compared to saline treated P5 pups (26mm 3 + SEM 6). Conclusion: This is the first study to show that TLR-3 is expressed in the immature brain and mediates preconditioning against ischemic injury. LPS-mediated TLR-4 activation is necessary for inducing preconditioning in P7 rats whereas poly I:C is effective in protecting P5 pups where TLR-3 expression predominates. Therefore, the role of TLRs in protecting the brain from ischemic injury is developmentally determined.