Effects of 9-t-butyl doxycycline on the innate immune response to CNS ischemia-reperfusion injury.

Cerebral ischemia triggers a cascade of neuroinflammatory and peripheral immune responses that contribute to post-ischemic reperfusion injury. Prior work conducted in CNS ischemia models underscore the potential to harness non-antibiotic properties of tetracycline antibiotics for therapeutic benefit. In the present study, we explored the immunomodulatory effects of the tetracycline derivative 9-tert-butyl doxycycline (9-TB) in a mouse model of transient global ischemia that mimics immunologic aspects of the post-cardiac arrest syndrome. Pharmacokinetic studies performed in C57BL/6 mice demonstrate that within four hours after delivery, levels of 9-TB in the brain were 1.6 and 9.5-fold higher than those obtained using minocycline and doxycycline, respectively. Minocycline and 9-TB also dampened inflammation, measured by reduced TNFα-inducible, NF-κß-dependent luciferase activity in a microglial reporter line. Notably, daily 9-TB treatment following ischemia-reperfusion injury in vivo induced the retention of polymorphonuclear neutrophils (PMNs) within the spleen while simultaneously biasing CNS PMNs towards an anti-inflammatory (CD11bLowYm1+) phenotype. These studies indicate that aside from exhibiting enhanced CNS delivery, 9-TB alters both the trafficking and polarization of PMNs in the context of CNS ischemia-reperfusion injury.

Lung SOD3 limits neurovascular reperfusion injury and systemic immune activation following transient global cerebral ischemia.

BACKGROUND AND OBJECTIVES: Studies implicate the lung in moderating systemic immune activation via effects on circulating leukocytes. In this study, we investigated whether targeted expression of the antioxidant extracellular superoxide dismutase (SOD3) within the lung would influence post-ischemic peripheral neutrophil activation and CNS reperfusion injury. METHODS: Adult, male mice expressing human SOD3 within type II pneumocytes were subjected to 15 min of transient global cerebral ischemia. Three days post-reperfusion, lung and brain tissue was collected and analyzed by immunohistochemistry for inflammation and injury markers. In vitro motility and neurotoxicity assays were conducted to ascertain the direct effects of hSOD3 on PMN activation. Results were compared against C57BL/6 age and sex-matched controls. RESULTS: Relative to wild-type controls, hSOD3 heterozygous mice exhibited a reduction in lung inflammation, blood-brain barrier damage, and post-ischemic neuronal injury within the hippocampus and cortex. PMNs harvested from hSOD3 mice were also resistant to LPS priming, slower-moving, and less toxic to primary neuronal cultures. CONCLUSIONS: Constitutive, focal expression of hSOD3 is neuroprotective in a model of global cerebral ischemia-reperfusion injury. The underlying mechanism of SOD3-dependent protection is attributable in part to effects on the activation state and toxic potential of circulating neutrophils. These results implicate lung-brain coupling as a determinant of cerebral ischemia-reperfusion injury and highlight post-stroke lung inflammation as a potential therapeutic target in acute ischemic cerebrovascular injuries.

Characterization of neutrophil-neuronal co-cultures to investigate mechanisms of post-ischemic immune-mediated neurotoxicity.

BACKGROUND: Immune-mediated reperfusion injury is a critical component of post-ischemic central nervous system (CNS) damage. In this context, the activation and recruitment of polymorphonuclear neutrophils (PMNs) to the CNS induces neurotoxicity in part through the release of degradative enzymes, cytokines, and reactive oxygen species. However, the extent to which close-range interactions between PMNs and neurons contribute to injury in this context has not been directly investigated. NEW METHOD: We devised a co-culture model to investigate mechanisms of PMN-dependent neurotoxicity. Specifically, we established the effect of PMN dose, co-incident neuronal ischemia, lipopolysaccharide (LPS)-induced PMN priming, and the requirement for cell-cell contact on cumulative neuron damage. RESULTS AND COMPARISON TO EXISTING METHOD(S): Pre-exposure of day in vitro 10 primary cortical neurons to oxygen-glucose deprivation (OGD) enhanced PMN-dependent neuronal death. Likewise, LPS-induced priming of the PMN donor further increased PMN-induced toxicity in vitro compared to saline-injected controls. Compartmentalization of LPS-primed PMNs using net wells confirmed the requirement for close-range cell-cell interactions in the process of PMN-induced neuronal injury. Moreover, time-lapse imaging and quantitative neurite analyses implicate PMN-neurite interactions in this pathological response. These experiments establish a platform to investigate immune and neural factors that contribute to post-ischemic neurodegeneration. CONCLUSIONS: Ischemic and immune priming enhance neurotoxicity in PMN-neuronal co-cultures. Moreover, cell-cell contact and neurite destruction are prominent features in the observed mechanism of post-ischemic neuronal death.

Lung-Derived SOD3 Attenuates Neurovascular Injury After Transient Global Cerebral Ischemia.

Background Systemic innate immune priming is a recognized sequela of post-ischemic neuroinflammation and contributor to delayed neurodegeneration. Given mounting evidence linking acute stroke with reactive lung inflammation, we asked whether enhanced expression of the endogenous antioxidant extracellular superoxide dismutase 3 (SOD3) produced by alveolar type II pneumocytes would protect the lung from transient global cerebral ischemia and the brain from the delayed effects of ischemia-reperfusion. Methods and Results Following 15 minutes of global cerebral ischemia or sham conditions, transgenic SOD3 and wild-type mice were followed daily for changes in weight, core temperature, and neurological function. Three days after reperfusion, arterial and venous samples were collected for complete blood counts, flow cytometry, and SOD3 protein blotting, and immunohistochemistry was performed on lung and brain tissue to assess tissue injury, blood-brain barrier permeability, and neutrophil transmigration. Relative to ischemic controls, transgenic SOD3 mice performed better on functional testing and exhibited reduced peripheral neutrophil activation, lung inflammation, and blood-brain barrier leak. Once released from the lung, SOD3 was predominantly not cell associated and depleted in the venous phase of circulation. Conclusions In addition to reducing the local inflammatory response to cerebral ischemia, targeted enrichment of SOD3 within the lung confers distal neuroprotection against ischemia-reperfusion injury. These data suggest that therapies geared toward enhancing adaptive lung-neurovascular coupling may improve outcomes following acute stroke and cardiac arrest.