RESUMO
Background: Necrotizing enterocolitis (NEC) is an inflammatory gastrointestinal process that afflicts approximately 10% of preterm infants born in the United States each year, with a mortality rate of 30%. NEC severity is graded using Bell's classification system, from stage I mild NEC to stage III severe NEC. Over half of NEC survivors present with neurodevelopmental impairment during adolescence, a long-term complication that is poorly understood but can occur even after mild NEC. Although multiple animal models exist, none allow the experimenter to control nor represent the gradient of symptom severities seen in NEC patients. We bridge this knowledge gap by developing a graded murine model of NEC and studying its relationship with neuroinflammation across a range of NEC severities. Methods: Postnatal day 3 (P3) C57BL/6 mice were fed a formula containing different concentrations (0% control, 0.25%, 1%, 2%, and 3%) of dextran sodium sulfate (DSS). P3 mice were fed every 3 hours for 72-hours. We collected data on weight gain and behavior (activity, response, body color) during feeding. At the end of the experiment, we collected tissues (intestine, liver, plasma, brain) for immunohistochemistry, immunofluorescence, and cytokine and chemokine analysis. Results: Throughout NEC induction, mice fed higher concentrations of DSS died sooner, lost weight faster, and became sick or lethargic earlier. Intestinal characteristics (dilation, color, friability) were worse in mice fed with higher DSS concentrations. Histology revealed small intestinal disarray among mice fed all DSS concentrations, while higher DSS concentrations resulted in reduced small intestinal cellular proliferation and increased hepatic and systemic inflammation. In the brain, IL-2, G-CSF, and CXCL1 concentrations increased with higher DSS concentrations. Although the number of neurons and microglia in the CA1 hippocampal region did not differ, microglial branching was significantly reduced in DSS-fed mice. Conclusion: We characterize a novel graded model of NEC that recapitulates the full range of NEC severities. We show that mild NEC is sufficient to initiate neuroinflammation and microglia activation. This model will facilitate studies on the neurodevelopmental effects of NEC.
RESUMO
To minimize the neurotoxic injury by clot-derived substances after intracerebral hemorrhage (ICH) on the surrounding brain tissue, minimally invasive neurosurgical protocols have evolved evacuating the hematoma by stereotaxic injection of a fibrinolytic agent such as recombinant tissue plasminogen activator (rtPA), followed by aspiration of the lysed clot. However, the possible contribution of the presence of exogenous tPA itself to the toxic effects of hematoma-derived factors complicates the rationale and efficacy of this therapeutic approach. To clarify the role of exogenous rtPA on edema development, we examined the extent of edema formation in a murine model of collagenase-induced ICH, which included tPA-deficient (tPA-/-) and wild-type (wt) mice. In 16 (7 tPA-/- and 9 wt mice) out of 32 mice, 1 mg/kg rtPA was injected into the hematoma 5 h after ICH induction followed by aspiration of the liquefied clot 20 min later. In the control group (8 tPA-/- and 8 wt mice), only collagenase was injected. The edema volume was quantified using SPOT software on Luxol Fast Blue and Cresyl violet-stained cross-sections 24 h, 3, and 7 days post surgery. Twenty-four hours after ICH induction, tPA-/- mice had a significantly smaller edema volume (P< 0.01), even when rtPA was administered. Between days 3 and 7 after ICH, exogenous rtPA exerts its edema-promoting effect irrespective of the underlying genotype and exhibits an extensive microglial activation adjacent to the clot. In conclusion, the role of the endogenous tPA appears to be limited to the early phase of edema formation, whereas exogenous rtPA is edema-promoting between days 3 and 7 after ICH.
