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  • TLR-4-Regulated Cerebrospinal Fluid Hypersecretion in Post-Hemorrhagic Hydrocephalus

    Final Number:

    Jason K. Karimy M.S.; Jinwei Zhang; David B Kurland BA; Brianna Carusillo Theriault PhD; Daniel Duran MD; Charuta Gavankar Furey BA; Volodymyr Gerzanich MD, PhD; J. Marc Simard MD; Kristopher Thomas Kahle MD PhD

    Study Design:
    Laboratory Investigation

    Subject Category:

    Meeting: Congress of Neurological Surgeons 2017 Annual Meeting

    Introduction: Intraventricular hemorrhage (IVH) frequently causes post-hemorrhagic hydrocephalus (PHH) as a result of impaired cerebrospinal fluid (CSF) homeostasis.[1,2] The most common treatment for PHH is surgical CSF shunting, a procedure fraught with complications.[3,4] Historically, it is thought that PHH results from impaired reabsorption of CSF, however, little consideration has been given to the role of CSF hypersecretion. Recently, toll-like receptor-4 (TLR-4) has been implicated in the CNS by detecting “alarmins”,[5] including IVH-derived metabolites,[6] via recognition of damage-associated molecular patterns.[7,8] We speculate that IVH triggers TLR-4-dependent inflammation at the choroid plexus epithelia (CPE), leading to CSF hypersecretion contributing to the development of PHH.

    Methods: In an established rat model of IVH,[9] we assessed the rate of CSF secretion, CPE inflammation and the effect of genetic and pharmacological intervention on the development of PHH. We developed and implemented a novel surgical method in rats to measure the rate of CSF secretion.[10] TLR-4 knockout rats,[11] antisense oligodeoxynucleotide-mediated gene knockdown,[12] and intracerebroventricular delivery of FDA-approved drugs were used to assess the role of specific inflammatory and ion transport targets on CSF secretion rate and ventriculomegaly following IVH. Immunoblot and immunohistochemistry were used to detect and quantify inflammatory markers.

    Results: We show IVH triggers TLR4-NFkB-mediated inflammation at the CPE that is associated with a striking ~3-fold increase in CSF secretion sufficient to cause PHH. CSF hypersecretion is dependent on the NFkB-regulated kinase SPAK,[13-15] which interacts with and phosphorylates the bumetanide-sensitive NKCC1 at the CPE apical membrane. Genetic ablation of TLR-4 or SPAK, and pharmacology antagonizing TLR-4-NFkB signaling or the SPAK-NKCC1 complex normalizes CSF secretion rates and ventriculomegaly after IVH.

    Conclusions: These data uncover a previously unrecognized role for CSF hypersecretion in the pathogenesis of PHH, and reveal a novel TLR-4-dependent regulatory pathway of CSF secretion that can be targeted with repurposed, FDA-approved drugs for the non-surgical treatment of PHH.

    Patient Care: We demonstrate a connection between PHH and CSF hypersecretion in the context of an innovative damage-associated signaling cascade in the CPE. Elucidating the molecular mechanisms involved in this cascade has revealed novel, actionable targets for pharmacotherapy and other alternative treatment options. With this study, we intend to improve the current standard of care for patients with PHH, offering new, targeted therapeutic alternatives that may reduce or bypass the need for shunt placement as a first-line measure.

    Learning Objectives: By the conclusion of this session, participants should be able to 1) Describe the importance of elucidating the molecular mechanisms underlying PHH; 2) Discuss in small groups the role of CSF hypersecretion as a contributing factor in the formation of PHH 3) Identify potentially effective treatment targets in the inflammation-dependent signaling cascade involved in PHH.

    References: 1. Cherian, S., Whitelaw, A., Thoresen, M. & Love, S. The pathogenesis of neonatal post-hemorrhagic hydrocephalus. Brain Pathol 14, 305-311 (2004). 2. Strahle, J., et al. Mechanisms of hydrocephalus after neonatal and adult intraventricular hemorrhage. Transl. Stroke Res 3, 25-38 (2012). 3. Paulsen, A.H., Lundar, T. & Lindegaard, K.F. Twenty-year outcome in young adults with childhood hydrocephalus: assessment of surgical outcome, work participation, and health-related quality of life. Journal of neurosurgery. Pediatrics 6, 527-535 (2010). 4. Paulsen, A.H., Lundar, T. & Lindegaard, K.F. Pediatric hydrocephalus: 40-year outcomes in 128 hydrocephalic patients treated with shunts during childhood. Assessment of surgical outcome, work participation, and health-related quality of life. Journal of neurosurgery. Pediatrics 16, 633-641 (2015). 5. Tewary, P., et al. Granulysin activates antigen-presenting cells through TLR4 and acts as an immune alarmin. Blood 116, 3465-3474 (2010). 6. Kwon, M.S., et al. Methemoglobin is an endogenous toll-like receptor 4 ligand-relevance to subarachnoid hemorrhage. International journal of molecular sciences 16, 5028-5046 (2015). 7. Belcher, J.D., et al. Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease. Blood 123, 377-390 (2014). 8. Shi, H., et al. TLR4 links innate immunity and fatty acid-induced insulin resistance. The Journal of clinical investigation 116, 3015-3025 (2006). 9. Simard, P.F., et al. Inflammation of the choroid plexus and ependymal layer of the ventricle following intraventricular hemorrhage. Transl. Stroke Res 2, 227-231 (2011). 10. Karimy, J.K., et al. A novel method to study cerebrospinal fluid dynamics in rats. J. Neurosci. Methods 241, 78-84 (2015). 11. Ferguson, C., McKay, M., Harris, R.A. & Homanics, G.E. Toll-like receptor 4 (Tlr4) knockout rats produced by transcriptional activator-like effector nuclease (TALEN)-mediated gene inactivation. Alcohol (Fayetteville, N.Y.) 47, 595-599 (2013). 12. Nugent, B.M., Valenzuela, C.V., Simons, T.J. & McCarthy, M.M. Kinases SPAK and OSR1 are upregulated by estradiol and activate NKCC1 in the developing hypothalamus. The Journal of neuroscience : the official journal of the Society for Neuroscience 32, 593-598 (2012). 13. Alessi, D.R., et al. The WNK-SPAK/OSR1 pathway: master regulator of cation-chloride cotransporters. Science signaling 7, re3 (2014). 14. Yan, Y., et al. Nuclear factor-kappaB is a critical mediator of Ste20-like proline-/alanine-rich kinase regulation in intestinal inflammation. The American journal of pathology 173, 1013-1028 (2008). 15. Yan, Y. & Merlin, D. Ste20-related proline/alanine-rich kinase: a novel regulator of intestinal inflammation. World journal of gastroenterology 14, 6115-6121 (2008).

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