Germinal matrix hemorrhage (GMH) is defined as the rupture of immature blood vessels within the subependymal brain tissue. Occurring in approximately 3.5 per 1,000 births, GMH presents a leading cause of mortality and morbidity in premature infants. Debilitating consequences of GMH include the formation of post- hemorrhagic hydrocephalus, leading to brain atrophy and neurological impairments. A major causative factor of hydrocephalus formation is thrombin, a coagulation factor, activated by the intracranial bleed. Thrombin initiates inflammatory responses, gliosis and overproduction of extracellular matrix (ECM) proteins, which obstruct the cerebroventricular system and impair CSF drainage. Thrombin participates in the proliferation of scar tissue by activating a subfamily of G protein-coupled receptors, named proteinase-activated-receptors (PARs). Once stimulated, PARs will activate mTOR, which has been reported to induce overproduction of ECM proteins, thus resulting in obstruction and impaired CSF drainage. Our first corollary hypothesis is that by blocking PARs and their downstream targets, hydrocephalus will be reduced after GMH. Thrombin will also lead to the formation of peri- and intraventricular blood clots, which mechanically impair the circulation and absorption of CSF, thus leading to hydrocephalus formation after GMH. Our second corollary hypothesis is that enhancing blood clot resolution and clearance, via macrophage activation, will effectively reduce hydrocephalus and consequent neurological deficits after GMH. We will implement pharmacological activation of peroxisome proliferator-activated receptor gamma (PPAR-?), which has been reported to increase microglial phagocytosis of red blood cells, thus decreasing residual clot sizes. From existing literature on adult intracerebral hemorrhage and from our own preliminary observations after experimental GMH, we propose to characterize the extent of GMH-induced brain injury and provide novel non- invasive therapeutic strategies. Our central hypothesis is that targeting thrombin downstream effectors (PARs &mTOR) and clot clearance (via PPAR-?) will reduce GMH-induced hydrocephalus and improve long term neurological function in this neonatal GMH rat model. We will evaluate the implication of PARs and PPAR-? with respect to GMH pathology and therapy in the following aims:
Aim 1 will investigate the role of thrombin and clot formation in post-hemorrhagic hydrocephalus in a novel GMH rat model. We hypothesize that GMH blood clots will impair the CSF circulation and an increase of thrombin activity will promote extracellular matrix proliferation, leading to disturbances in normal CSF dynamics and the development of hydrocephalus and long-term neurological deficits.
Aim 2 will determine the role of thrombin downstream effectors (PARs &mTOR) in GMH induced hydrocephalus. We hypothesize that the activation of PARs by thrombin will cause the overproduction of extracellular matrix proteins, via mTOR activation, thus obstructing CSF drainage and inducing hydrocephalus. PAR inhibition will reduce extracellular matrix proliferation and hydrocephalus.
Aim 3 will determine the role of PPAR-? in clot clearance after GMH. We hypothesize that PPAR-? activation will activate microglial phagocytosis of red blood cells, hence reducing blood clots and hydrocephalus. The long-term goals of this proposal are to provide non-invasive therapeutic approaches for GMH patients.

Public Health Relevance

One in every 300 newborn babies in the United States will have bleeding inside the brain tissues during or immediately after labor. This bleeding may either kill or cause lifelong disabilities that will not only affect the baby but also impact the lves of parents, and communities that raise, teach and care for them. This proposal will answer why bleeding in the brain causes water accumulation (hydrocephalus) which damages surrounding brain tissues and leads to long term disability. Current surgical treatment (shunting) is invasive and ineffective. Our proposed non-invasive pharmacological treatment strategies if proved beneficial, will potentially impact the clinical management of this devastating condition.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Brain Injury and Neurovascular Pathologies Study Section (BINP)
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Koenig, James I
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Loma Linda University
Other Basic Sciences
Schools of Medicine
Loma Linda
United States
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Ma, Li; Manaenko, Anatol; Ou, Yi-Bo et al. (2017) Bosutinib Attenuates Inflammation via Inhibiting Salt-Inducible Kinases in Experimental Model of Intracerebral Hemorrhage on Mice. Stroke 48:3108-3116
Rolland, William B; Krafft, Paul R; Lekic, Tim et al. (2017) Fingolimod confers neuroprotection through activation of Rac1 after experimental germinal matrix hemorrhage in rat pups. J Neurochem 140:776-786
Lekic, Tim; Klebe, Damon; Flores, Jerry et al. (2016) Remote Ischemic Postconditioning (RIPC) After GMH in Rodents. Acta Neurochir Suppl 121:63-7
Malaguit, Jay; Casel, Darlene; Dixon, Brandon et al. (2016) Intranasal Osteopontin for Rodent Germinal Matrix Hemorrhage. Acta Neurochir Suppl 121:217-20
Flores, Jerry J; Klebe, Damon; Rolland, William B et al. (2016) PPAR?-induced upregulation of CD36 enhances hematoma resolution and attenuates long-term neurological deficits after germinal matrix hemorrhage in neonatal rats. Neurobiol Dis 87:124-33
Lekic, Tim; Krafft, Paul R; Klebe, Damon et al. (2016) Cyclooxygenase-2 Inhibition Provides Lasting Protection Following Germinal Matrix Hemorrhage in Premature Infant Rats. Acta Neurochir Suppl 121:203-7
Lekic, Tim; Flores, Jerry; Klebe, Damon et al. (2016) Intranasal IGF-1 Reduced Rat Pup Germinal Matrix Hemorrhage. Acta Neurochir Suppl 121:209-12
Shi, Xudan; Doycheva, Desislava Met; Xu, Liang et al. (2016) Sestrin2 induced by hypoxia inducible factor1 alpha protects the blood-brain barrier via inhibiting VEGF after severe hypoxic-ischemic injury in neonatal rats. Neurobiol Dis 95:111-21
Lekic, Tim; Zhang, John H (2016) Exsanguination Postconditioning of ICH (EPIC-H) Using the Lancet for Brain Bleed in Rodents, Preliminary Study. Acta Neurochir Suppl 121:49-53
Lekic, Tim; Krafft, Paul R; Klebe, Damon et al. (2016) PAR-1, -4, and the mTOR Pathway Following Germinal Matrix Hemorrhage. Acta Neurochir Suppl 121:213-6

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