Background & Aims
Newborns admitted to the neonatal intensive care unit are exposed to an average of 10-14 painful diagnostic procedures daily, with the most common procedure being the heel stick. During this critical period, the newborn’s central nervous system (CNS) is readily influenced by noxious stimuli resulting in altered neuroplasticity and immature descending inhibitory mechanisms. Downstream these changes can increase the risk for the newborn to develop cognitive/motor developmental delays, autism spectrum disorder, Alzheimer disease, and amyotrophic lateral sclerosis. To our knowledge, no study has explored the behavioral implications of early life injury into early adulthood. Here we aim to investigate alterations in spinal microglia activation, cytokine expression, and identify phenotypic alterations in behavior following early life injury. We hypothesize that repetitive early life injury will increase microglia activation, elicit expression of targeted cytokines, and impact neurobehavior.
Methods
Sprague Dawley rats (n= 153) were bred in house and randomized based on sex into one of three experimental groups: repetitive needle prick (RNP) to mimic clinically relevant heel stick, non-noxious tactile stimulation (TS), or unmanipulated control (UC) groups. Animals from each experimental group were further randomized into 1 of 6 sacrifice timepoints (1, 2, 4, 8, 12, or 17-weeks of life). During each timepoint, rats were perfused using 4% paraformaldehyde and the lumbar spinal cord was extracted and stored for evaluation. Immunohistochemistry was used to assess alterations in microglia and cytokine expression and microglia morphological adaptations. Additionally, rats with later sacrifice timepoints (8 to 17-weeks of life) underwent a battery of behavioral and physical assessments to detect changes in quantitative somatosensory sensitivities, non-reflexive limb-use, and exploratory/anxiety behavior.
Results
RNP animals demonstrated a significant peaked increase in the total quantity of spinal microglia at 1-week (p= <0.0001, <0.0001), mean intensity at 8-weeks (p=0.0004, <0.0001), cell body volume at 8-weeks (p= 0.0001, 0.0006), and projection area at 8-weeks (p= 0.0016, 0.0473) when compared to TS and UC, respectively; while significant decreases in spinal microglia spheric shape was observed at 4-weeks (p=0.0003, 0.0017). Additionally, RNP displayed a significant peak increase in mean expression of TNF-? (p=0.0106, 0.0043) at 2-week, IL-6 (p= 0.0004, <0.0001) at 1-week, and IL-10 (p= <0.0001, <0.0001) at 1-week. Although a small increase in spinal microglia mean intensity was observed between 4 through 12-weeks of life in TS, these findings were not significant when compared to UC and remained significantly lower when compared to RNP. Overall, no differences in behavior outcomes were detected. No sex differences were detected.
Conclusions
Overall, this study suggests that RNP may play a role in stimulating a CNS inflammatory chain reaction when compared to both TS and UC. RNP during the first 7-days of life led to a morphological and physiological transformation of spinal microglia from resting to phagocytic state throughout the first 17-weeks of life. RNP also led to an increase in cytokine expression in TNF-?, IL-6, and IL-10. Interestingly, no phenotypic alterations in behavior or somatosensory sensitivity were detected in animals that received RNP. Altogether, our study indicates that RNP could play a significant role in stress/trauma-induced inflammation within the CNS without impacting the specific neurobehaviors assessed. Future studies are needed to explore the relationship that early life altered microglia and cytokine expression play in the development of neurogenerative disorders and neurodevelopmental. These studies may lay a solid foundation for the discovery of future novel therapeutic targets/biomarkers.
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Presenting Author
Jeannette Delva-Wiley
Poster Authors
Topics
- Pain in Special Populations: Infants/Children