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  • Heme Induces Microglial CXCL2 Release - a Mechanism of Neutrophil-Mediated Injury after Intracerebral Hemorrhage

    Final Number:

    David B Kurland BA; Volodymyr Gerzanich MD, PhD; J. Marc Simard MD

    Study Design:
    Laboratory Investigation

    Subject Category:

    Meeting: Congress of Neurological Surgeons 2013 Annual Meeting

    Introduction: Neutrophils recruited into the CNS after intracerebral hemorrhage (ICH) are an important source of secondary injury. Chemokines are released following ICH, which act as chemoattractants for neutrophils and other inflammatory cells. In the setting of ICH in rodents, the neutrophil-specific chemokine CXCL2 is upregulated and is associated with severity of injury. CXCL2 signals via interaction with CXCR2, the same binding partner of CXCL8/IL-8, the most important neutrophil chemokine in humans. Heme, present in high concentrations locally after ICH, has recently been characterized as an endogenous ligand for Toll-like Receptor 4 (TLR4). TLR4 is well-known as the receptor for LPS and is critical in initiating the innate inflammatory response to bacterial infection. Additionally, TLR4 activation is upstream of CXCL2 production.

    Methods: Intrastriatal injection of whole blood, heme or CXCL2 was performed under stereotactic guidanace. Isolation of resting adult microglia was performed using percoll gradients. An ELISA kit was utilized in the measurement of CXCL2 in cell culture supernatant. Behavioral testing was recorded using the 5-Choice-Serial-Reaction-Time-Test apparatus. Standard immunolabeling procedures for CXCL2 and TLR4 were employed.

    Results: Here we report results from in vivo experiments showing that physiological concentrations of heme lead to upregulation of CXCL2 by IHC. Furthermore, in cultures of freshly isolated microglia from adult rat hippocampus, application of heme results in significant release of CXCL2 as measured by ELISA. Finally, intrastriatal injection of CXCL2 leads to acute accumulation of neutrophils and produces complex neurobehavioral deficits in rats trained in the 5-Choice Serial Reaction Time task.

    Conclusions: We conclude that microglial release of CXCL2 may play a role in neutrophil-mediated injury after ICH. Therapeutic intervention aimed at disrupting this chemokine signal may prove to be beneficial in treating this devastating injury.

    Patient Care: Intracerebral hemorrhage (ICH) is a devastating injury, with complex etiology and pathology. ICH occurs when a blood vessel ruptures within the brain parenchyma, and it accounts for 15% of strokes annually in the United States, roughly 20 cases per 100,000. While second to ischemic stroke in prevalence, data on morbidity and mortality illustrate the staggering severity of this type of injury. Thirty-day mortality is higher than 40% in some studies, and permanent neurological impairment is extremely common among survivors. Over the past several decades, significant progress has been made toward understanding the complexities of stroke, but translational research has been primarily focused on ischemic stroke. Up to now, no preclinical studies have led to a successful phase 3 clinical trial in intracerebral hemorrhage. In order to improve outcomes following hemorrhagic stroke, the unique mechanisms of injury induced by blood in the brain need to be better understood.

    Learning Objectives: By the conclusion of this session, participants should be able to: 1) Describe the importance of neutrophil-mediated injury following intracerebral hemorrhage, 2) Discuss, in small groups, the relevance of recruited leukocytes in the pathology of CNS injury, 3) Identify potential treatment options for hemorrhagic stroke.

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