University of Turku, Finland Turku, Varsinais-Suomi, Finland
Background: Neonatal hypoxic-ischemic encephalopathy (HIE) is considered the most severe birth complication related to perinatal oxygen deprivation. Insufficient oxygen and glucose delivery leads to a cascade of events where inflammation, excitotoxicity, and oxidative stress further exacerbate the tissue injury. Objective: A C57BL/6J HIE mouse model was constructed to characterize how leukocyte populations in the bloodstream and brain parenchyma are affected by hypoxic-ischemic injury. Design/Methods: 9-day-old C57BL/6J mouse pups underwent unilateral left common carotid artery ligation followed by exposure to systemic hypoxia (9% O2 and 91% N) for 60min to cause a unilateral HIE. The level of brain injury was confirmed a week after surgery by H&E and immunohistochemical stainings. Leukocytes were isolated from blood and homogenized brain samples at different time points and stained with fluorophore-conjugated primary antibodies for flow cytometric analysis. The phenotype of immune cells was measured and compared between control and HIE mice. In addition, the expression levels of multiple cytokines and chemokines will be compared using qPCR. Results: Immunohistological stainings revealed consistent morphological changes in the ipsilateral hypoxic-ischemic hemispheres, including severe hippocampal atrophy. In addition, anti-GFAP and anti-Iba-1 labeling revealed widespread ipsilateral activation of astrocytes and microglia in the striatum, hippocampus, and thalamus in the HIE mice. Flow cytometric analyses revealed a significantly increased frequency of CD4+ (p = 0.0184) and CD8+ (p = 0.0185) T-lymphocytes in the hypoxic-ischemic brain already at the 6h time point after the hypoxic-ischemic injury. At the 12h time point, the frequency of neutrophils (p = 0.0366) and B cells (p = 0.0366) grew significantly in the hypoxic-ischemic brain. 24h after hypoxic-ischemic injury, the frequency of resident microglia (p = 0.0362) and Ly6C high monocytes (p = 0.0165) were significantly higher in the hypoxic-ischemic brain. However, no significant changes were observed in the leukocyte populations at the 72h time point.
Conclusion(s): Flow cytometric analyses reveal important pathophysiological findings of the development of hypoxic-ischemic brain injury. Data gathered from these studies can be used to characterize further leukocyte subpopulations and test possible new biomarkers in the neonatal HIE.
