A traumatic impact acutely injures the brain by mechanical forces of shear strains and deformation in the form of a shockwave (e.g., as in a blast injury). Head trauma patients often suffer cognitive and psychological deficits without visible wounds. There is an unmet need for surrogate biochemical markers that could be measured in the patient's brain fluids to signal that an injury to the central nervous system had occurred. Such biomarkers would be helpful to diagnose patients rapidly and unambiguously and to prevent exposure to a potential catastrophic repeated impact. The work proposed here addresses this basic need for a biomarker of neural injury. By using a well characterized in vitro injury model, we will determine the protein release of human astrocytes to mechanical trauma and identify an astrocyte signature of the proteins that are found in trauma patient's cerebrospinal fluids (CSF). Astrocytes are the most abundant cell type in the brain, they respond to any kind of insult, support nerve cells and could therefore be considered as the 'bodyguards'of the brain. We will use human astrocytes for translational relevance. The simple mechanical trauma culture model uses a controlled pressure pulse that abruptly stretches astrocytes grown on deformable dishes and causes widespread and reproducible injuries. The mechanical insult leads to cell death and damage in the form of pores in the cells'membrane that can be repaired or cause death later on. In addition, the mechanical stimulus also activates astrocytes to become reactive. We will document their reactivity by showing activation of a transcription factor and documenting shape changes and enlargement using time-lapse movies, staining and quantitative imaging techniques. The cells release proteins into the fluids that we will analyze. Proteins that are markedly altered in the fluid of traumatized astrocytes will be identified by using a proteomic approach. We will also compare differences between protein profiles from cells that received a mild versus those that got a severe pressure pulse. The identified proteins and their profiles of change will be related to the extent of membrane damage and cell death that we also measure in the injured cells. Chosen candidates for biomarkers released from traumatized astrocytes in the cultures will then be detected in the CSF of head trauma patients using a unique mass spectrometry method. This quantitative analytical approach is new to the neurotrauma field and does not require the availability of antibodies. We will establish this approach to detect and quantify candidate trauma markers in injured and non-injured patients'brain fluids to show an astrocyte signature of neurotrauma in a fluid that is regularly sampled in the clinical setting to relieve head pressure. This work will help to set the technical groundwork for the future development of diagnostic tools for traumatic brain injury.
Neurotrauma encompasses patients with a range of traumatic brain and spinal cord injuries including soldiers with blast inflicted injury, infants with the often undiagnosed shaken baby syndrome and athletes exposed to repeated concussions but their wounds are often invisible in the clinic. There is an unmet need for reliable surrogate biochemical markers for neurotrauma to rapidly diagnose whether an injury to the central nervous system has occurred. This proposal addresses this need with a unique approach by combining a simple trauma model in a dish with protein-based screening tools that are new to the neurotrauma field and will provide new candidates for neurotrauma biomarkers that are released by astrocytes, the 'bodyguards of the brain'.
|Levine, Jaclynn; Kwon, Eunice; Paez, Pablo et al. (2016) Traumatically injured astrocytes release a proteomic signature modulated by STAT3-dependent cell survival. Glia 64:668-94|