Neutrophils are the most abundant leukocytes and usually the first immune cell-type recruited to a site of infection or tissue damage. In asphyxiated neonates, elevated peripheral neutrophil counts are associated with poorer neurological outcomes. Induced neutropenia provides brain protection in animal models of neonatal hypoxic-ischemic (HI) injury, but the anti-neutrophil serum used in past studies heavily cross-reacts with monocytes, thus complicating the interpretation of results. Here we examined neutrophil influx and extravasation, and used a specific anti-Ly6G antibody for induced neutropenia against lipopolysaccharide (LPS)-pretreated HI injury in murine neonates, a model for inflammation-sensitized hypoxic-ischemic encephalopathy (HIE). As early as 6 h after the LPS/HI insult, the mRNAs for neutrophil-recruiting and mitogenic chemokines ascended in the ipsilateral hemisphere, coinciding with immuno-detection of neutrophils. However, neutrophils mainly resided within blood vessels, exhibiting signs for neutrophil extracellular traps (NETs), before 48 h post-LPS/HI. Prophylactic anti-Ly6G treatment blocked the brain infiltration of neutrophils, but not monocytes or lymphocytes, and markedly decreased LPS/HI-induced pro-inflammatory cytokines, matrix metalloproteinase 9 (MMP-9), and brain tissue loss. In contrast, anti-Ly6G treatment at 4 h post-LPS/HI failed to prevent the influx of neutrophils and brain damage. Together, these results suggest important pathological functions for early-arriving neutrophils in inflammation-sensitized HIE.
Hypoxia-ischemia to brain triggers immune responses consisting of activation and recruitment of leukocytes and expression of pro-inflammatory mediators. Inducing neutropenia confers protection against cerebral ischemia in adult and neonatal rats, but it remains unclear if neutrophils enhances neonatal brain damage in inflammation (LPS)-sensitized hypoxic-ischemic (HI) insults to mice. Herein, we demonstrated that following LPS/HI insult, the mRNA expression of C-X-C motif ligands 1 and 2, two potent neutrophil chemoattractants, granulocyte colony-stimulating factor that stimulates the bone marrow to produce and release granulocytes, and pro-inflammatory cytokines, including interleukins 1β and 6 and tumor necrosis factor α, were increased in mouse brains. As early as six hours after insult, neutrophil infiltration was readily detectable in mouse brains and increased with time, especially in those brains with severe damage. Neutrophils either resided within vessel like structures or accumulated within brain parenchyma. In addition, infiltrated neutrophils expressed interleukin 1β and tumor necrosis factor α, as well as citrullinated histones, a marker of neutrophil extracellular traps, in the damaged brains. Importantly, prophylactic depletion of neutrophils from the periphery reduced leukocyte infiltration, the expression of pro-inflammatory mediators in mouse brains, and brain atrophy, whereas post-insult depletion of neutrophils conferred marginal protection. Collectively, our data support the pathogenic roles of neutrophils in the acute phase of inflammation-sensitized hypoxia-ischemia brain injury.
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