Traumatic brain injury (TBI) is a complex process encompassing three overlapping phases: a) primary injury, b) secondary injury and c) regenerative responses. Systemic administration of granulocyte-colony stimulating factor (G-CSF) represents a novel approach for reinforcing the brain's self-repair, especially during the secondary and regenerative phases.
The Specific Aims of this research program are designed to test the hypotheses that a) G-CSF indirectly enhances brain repair by promoting infiltration of bone marrow-derived cells (BMDC) to brain, modulate neuro-inflammatory processes and secrete trophic factors;b) G-CSF directly interacts with its neural cells receptors to trigger intra-cellular signaling cascades that decrease cell death and promote neurogenesis.
Aim 1 will determine the optimal dose of G-CSF and the recovery time-course of behavioral deficits after TBI in mice. Non-irradiated mice will be used to ensure the effects of G-CSF are not confounded by whole body irradiation and bone marrow transplantation (BMT). Motoric function (biased swing activity;rotarod) and behavioral (water maze) end-points will be assessed in mice at baseline, 3, 7, and 14 days after TBI. Secondary endpoints will measure a) lesion volume, b) extent of microgliosis and astrocytosis and c) brain regional levels of cytokines.
Aim 2 a. To assess the extent of BMDC mobilization triggered by TBI and modulated by G-CSF, the phenotypic fate and distribution of green fluorescent protein (GFP+) BMDC in chimeric mice brains will be determined using immunofluorescence to identify microglia, astrocytes, and neuron-like cells that co-express GFP. The time-course of infiltration of GFP+ BMDCs will be determined by assessing total GFP+ burden ipsilateral and contralateral to the TBI at 3, 7 and 14 days after injury.
Aim 2 b. To determine the extent to which BMDC penetration into brain is responsible for enhanced TBI recovery, the infiltration of BMDC into the central nervous system (CNS) will be attenuated or blocked with agents that block chemokine signaling to monocytes or utilization of mice with a knockout of the chemokine receptor CCR2. Enhanced recovery despite inhibition of BMDC mobilization will support the hypothesis that direct actions of G-CSF on neural cells play a major role.
Aim 3. To investigate the direct effects of G-CSF action on neural cells, the molecular impact of these cytokines on signal transduction, apoptosis and neurogenesis will be assessed in neural cell cultures. Results from this analysis will be compared to molecular analyses of signal transduction and anti- apoptosis in tissue samples dissected from TBI brains treated with G-CSF or vehicle. Methods: Chimeric mice will be generated that harbor GFP BMDCs to permit tracking the distribution and phenotypic fate of BMDCs that infiltrate the brain after TBI. Surgery: TBI will be delivered with a pneumatically driven controlled cortical impact (CCI) device to mice. Behavioral Assessments: Analyses of motor asymmetry (EBST), rotarod test and Water Maze (MWM). Endpoints: a) changes in behavior;b) changes in lesion volume;c) extent, distribution and phenotypic fate of GFP+ BMDC in brain assessed by double-labeling procedures;d) changes in cytokine profiles in brain regions;e) changes in signal transduction (PKC-?), Bcl2. Expected Results: G-CSF will modulate BMDCs infiltration and enhance recovery of behavioral deficits. Improvement of neurologic deficits will shown to be related to a combination of actions including a) changes in brain infiltration of BMDC;b) secretion of cytokines that promote neurogenesis;c) up-regulation of anti-apoptotic signaling triggered by G-CSF acting directly on its receptor in neural cells.
Military personnel serving in Iraq and Afghanistan and victims of terrorism-related attacks are at high risk of sustaining traumatic brain injury (TBI). Standard treatments are inadequate in light o the profound long-term disability documented in survivors of TBI. Novel approaches are necessary that reinforce the brain's natural self-repair response. G-CSF is a blood cell growth factor that stimulates generation of blood stem cells and white blood cells. This agent has been shown in animal experiments to enhance brain stem cell generation and to protect against brain cell death following stroke. Successful completion of this project in a mouse model of TBI will demonstrate that delivery of these agents will promote recovery from neurologic deficits. This project will provide support for translation to clinical studies in which G-CSF is administered within days of the injury to facilitate recovery from TBI.
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