Endotoxemia induces lung-brain coupling and multi-organ injury following cerebral ischemia-reperfusion

Abstract

Post-ischemic neurodegeneration remains the principal cause of mortality following cardiac resuscitation. Recent studies have implicated gastrointestinal ischemia in the sepsis-like response associated with the post-cardiac arrest syndrome (PCAS). However, the extent to which the resulting low-grade endotoxemia present in up to 86% of resuscitated patients affects cerebral ischemia-reperfusion injury has not been investigated. Here we report that a single injection of low-dose lipopolysaccharide (50μg/kg, IP) delivered after global cerebral ischemia (GCI) induces blood-brain barrier permeability, microglial activation, cortical injury, and functional decline in vivo, compared to ischemia alone. And while GCI was sufficient to induce neutrophil (PMN) activation and recruitment to the post-ischemic CNS, minimal endotoxemia exhibited synergistic effects on markers of systemic inflammation including PMN priming, lung damage, and PMN burden within the lung and other non-ischemic organs including the kidney and liver. Our findings predict that acute interventions geared towards blocking the effects of serologically occult endotoxemia in survivors of cardiac arrest will limit delayed neurodegeneration, multi-organ dysfunction and potentially other features of PCAS. This work also introduces lung-brain coupling as a novel therapeutic target with broad effects on innate immune priming and post-ischemic neurodegeneration following cardiac arrest and related cerebrovascular conditions.

Keywords: Blood-brain barrier; Endotoxin; Global cerebral ischemia; Ischemia-reperfusion injury; Microglia; Neurodegeneration; Neuroinflammation; Neutrophil; Post-cardiac arrest syndrome; Systemic inflammation.

Figure 1

LPS dose titration studies. IP administration of 50 μg/kg LPS yielded undetectable plasma endotoxin (A) and no changes in granulocyte count (B), core temperature (C), or weight (D), while effects were seen with 4 mg/kg and 20 mg/kg. (D) Elevated IL-6 was seen in all mice after 6 hours and remained elevated at 24 hours in mice receiving 20 mg/kg LPS. Values represent means ± SD (n = 3). 1-way ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Analyses of combined 3VO and systemic LPS on neurological function. (A) Following basilar artery (BA) occlusion, mice undergo either sham dissection or 15-minute common carotid artery (CCA) occlusion with saline or IP LPS injection immediately following reperfusion. Effects of experimental procedures on weight (B) and core temperature (C). (D) Effects of experimental procedures on neurological functional scores assessed prior to BAO (Day −10), prior to BCCAO/sham (Day 0), at Day 1, and at Day 3. Values represent means ± SD (n = 3–4 per group). 3-way ANOVA: ^† ANOVA main effects, * Dunn-Sidak multiple comparisons. *^/† p < 0.05, **^/†† p < 0.01, ^††† p < 0.001, ****^{/††††} p < 0.0001.

Figure 3

Effects of systemic LPS exposure on ischemia-induced cortical injury. (A) IHC of coronal brain sections demonstrates combined effects of 3VO/LPS treatment on dendritic injury (beading, loss of MAP2 staining; red) and neuron death (Fluoro-Jade C; green) on cortex (top) and CA1 of the hippocampus (bottom); scale bars = 100 μm. White dashed line outlines area of MAP2(−) cortex. (B) Histogram illustrating exacerbated injury caused by the addition of LPS after 3VO. % Injured cortex = MAP2(−) area, as outlined in (A, top), over total cortical area. (C) Combined 3VO and endotoxemia causes PECAM-1 upregulation (red) and parenchymal IgG deposition (green; white arrowheads); scale bar = 100 μm. 3VO/LPS treatment upregulates vascular PECAM-1 expression (D) and IgG deposition per vessel length (E) in 3VO/LPS animals. Values represent means ± SD (n = 3–4 per group). AU = arbitrary units. 2-way ANOVA: * Sham vs. 3VO; ^# SAL vs. LPS. ^## p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

IP LPS treatment enhances PMN accumulation in post-ischemic cortex at Day 3. IHC of cortical sections (A) and histogram (B) demonstrating Ly-6B(+) PMN infiltration after ischemia-reperfusion; scale bar = 200 μm. 2-way ANOVA: * Sham vs. 3VO; ^# SAL vs. LPS. * p < 0.05, ^## p < 0.01, **** p < 0.0001. (C) Correlation between PMN infiltration and cortical injury; Pearson’s r = 0.8177, p = 0.0002. Inverse relationship between PMN infiltration and Neurological Function Score; Pearson’s r = −0.8877, p < 0.0001. Values represent means ± SD (n = 3–4 per group).

Figure 5

Delayed effects of systemic LPS and PMN migration on neuroinflammation. (A) Immunohistochemical analyses of microglial activation (Iba1; red) and cortical PMN (Ly-6B; green) migration 3 days following treatment; scale bar = 100 μm. (B) Stimulatory effect of systemic LPS treatment on microglial activation after GCI assessed using average Iba1 fluorescence intensity per mm² field. 2-way ANOVA: * Sham vs. 3VO; ^# SAL vs. LPS. ** p < 0.01, ****^/#### p < 0.0001. (C) Representative heat maps illustrating differences in microglial arborization across treatment groups. Heat maps are cell-specific with red indicating the radius with the greatest number intersections; scale bar = 15 μm. (D) Sholl analysis of microglia in 3VO/SAL vs. 3VO/LPS animals. (E) Sholl analysis demonstrating blunted arborization in microglia in PMN-rich cortex of 3VO/LPS mice. Values represent means ± SD (n = 3–4 per group). Repeated measures ANOVA: * p < 0.05, NS = no significant difference between PMN(+) and PMN(−) cortex. Values represent means ± SD (n = 3–4 per group).

Figure 6

Timing of peripheral neutrophil priming in the GCI model. Flow cytometry analyses demonstrating the effects of systemic inflammation (Sham/LPS), GCI (3VO/SAL), and combined treatment (3VO/LPS) on CD11b expression on peripheral Ly-6G^Hi/CD11b^Hi PMNs 2, 4, and 6 hours after reperfusion compared to Sham/SAL-treated mice. Values represent 10,000 events per time point per group.

Figure 7

LPS enhances PMN accumulation in peripheral organs after GCI. Lung IHC (A) and quantitative analyses for average fluorescence intensity of Ly-6B(+) PMNs per mm² field (B) and alveolar wall thickness (C). PMN accumulation is observed in the liver (D–E) and kidney (F–G) with 3VO/LPS treatment but not with 3VO/SAL. Scale bars = 200 μm. Values represent means ± SD (n = 3–4 per group). AU = arbitrary units. 2-way ANOVA: * Sham vs. 3VO; ^# SAL vs. LPS. *^/# p < 0.05, **^/## p < 0.01, *** p < 0.001.