Poor neurodevelopmental outcomes continue to be a major problem for survivors of very premature birth (< 80% gestation). The fetal inflammatory response hypothesis postulates that endogenous fetal cytokines initiate early premature labor and are involved in injury to the developing CNS. Elevation of Interleukin (IL)-6 in fetal membranes and cord blood is one biomarker of fetal inflammatory response, predicts acute neuropathology and has been correlated with later structural and functional outcomes. In addition to its role in inflammation, IL-6 is known to be expressed at different times by brain region during CNS development, leading to the question of its potential role in neurobehavioral outcomes of the premature when IL-6 is expressed at an aberrant time.
Objective: To determine if exposure of the developing cerebral cortex to IL-6, absent traumatic, ischemic, or infectious insults, altered functional landmarks of nervous system development in a mouse model temporally analogous to human brain development at 24-28 weeks gestation. Design: Two-group longitudinal experimental design, with cross-sectional and repeated measures. Mice were anesthetized and injected sub-cranially with saline or Hyper-IL-6 (recombinant IL-6 attached to its soluble receptor) at post-natal day (pnd) 4 with prospective random assignment to endpoints at pnd 5, 11, 16, 24, and adulthood.
Sample: Twelve cross-fostered litters of 10 male cd-1 mice from in-house breeding colony. Outcome Variables: Acute tissue injury, physical and reflex development trajectory in pre-weanling pups; behavior, learning and memory in adults.
Methods: Acute tissue injury was measured at pnd 5 by immunohistochemistry and RNase protection assay for markers of necrosis and gliosis. Growth, development, and behavioral ontogeny were monitored until weaning (modified Fox Battery). At weaning and in adulthood, functional neurobehavioral outcomes were assessed (Functional Observation Battery, US EPA). Learning and memory was tested by passive avoidance at weaning, and by habituation and two radial arm maze paradigms in the adult. Cross-sectional data were analyzed by ANOVA, student’s t, or Kruskall-Wallace (SPSS). Within-subject repeated measures data were analyzed using Mixed General Linear Model (SAS).
Findings: No acute injury response was evident at 1-7 days following hyper-IL-6 injection as determined by cellularity and glial morphology and RNase protection assays for acute response genes, ICAM-1, iNOS, Mac-1, A-20, and EB-22. Physical and reflex development was unaltered in pre-weanling mice. However, exposed mice demonstrated sensorimotor alterations of over-generalized responses (p<.0001), slower cliff aversion, earlier walking, increased rearing and jumping (all p<.05, Fox Battery) leading to an overall impression of hyper-reactivity and hyperactivity. At pnd 80, assessment on the Functional Observation Battery showed persistence of hyper-reactivity and hyperactivity (general excitability domain, p<.001; sensory domain, p<.005). Learning and memory were assessed by passive avoidance (PA) at pnd 24 and by radial arm maze (RAM) in adults. Hyper-IL-6 exposure significantly impaired PA performance (p<.05, one tailed). RAM performance was similar between groups in acquisition of a win-shift task but hyper-IL-6 animals showed deficits on a repeated acquisition task.
Conclusion: The persistent hyperactive, hyper-reactive behavioral phenotype evident in hyper-IL-6 exposed mice appeared to interfere with performance on both learning and memory tasks.
Implications: These data suggest that early exposure of the developing cortex to IL-6 can have persistent adverse effects on neurobehavioral function. The findings lend support to the fetal inflammatory response hypothesis and raise additional concerns for effects of endogenous immune mediators on the developing CNS of very premature infants.
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