Hill laboratory focuses on spinal cord injury (SCI) repair and the development of therapeutic interventions to promote recovery of function. Studies range from examining the basic biology of axonal regeneration to testing small molecules and cellular transplants for their ability to promote axonal regeneration and functional recovery in rodent models of spinal cord injury.
Injury to the spinal cord causes direct damage to the cells within the spinal cord. This damage, referred to as the primary injury, results in the activation of biochemical cascades which results in damage to adjacent cells that were initially undamaged. This spread of damage is referred to as the secondary injury. Over the subsequent days to weeks, the injury site undergoes endogenous changes. The balance of the detrimental changes associated with the primary and secondary injury and the beneficial and detrimental changes associated with the endogenous wound healing response ultimately determines the amount of function retained.
Therapies for SCI focus on different aspects of the cellular and biochemical changes that occur. Early interventions focus on minimizing the spread of tissue damage or altering the progression of the cellular changes in order to preserve as much tissue and function as possible. In the lab, we test promising pharmacological interventions to promote and restore function using preclinical SCI models. To assess the outcomes of treatment, we use in vivo imaging, behavioral testing (e.g., open field locomotion, ladderwalk, gait analysis, and thermal and pressure sensation), histology (e.g., cryosectioning, immunohistochemistry, epifluorescence and confocal microscopy), biochemistry (e.g., Western blotting), and molecular biology (e.g., quantitative PCR). Many of these same outcome measures are also used when we examine the temporal changes in cells that occur following SCI. For these studies, we are identifying additional cellular changes that occur following SCI in order to identify and develop new therapeutic interventions.
SCI results in the disconnection of circuitry necessary for motor and sensory function. Cellular transplants can assist in preserving tissue and/or restoring circuitry. Several cell types are currently in Phase 1 clinical trials for SCI repair and their use for human SCI therapies appears to be safe. A variety of different cell and tissue types have been transplanted into spinal cord injury models (e.g., peripheral nerves, Schwann cells, olfactory ensheathing glia, fetal tissue, neural stem/progenitor cells, induced pluripotent cells). The specific effects of a given cell type differ, but in general cell transplants can release growth enhancing factors to preserve tissue, provide a substrate for axons to grow on, replace damaged neurons to establish network relays, and/or replace damaged glia to ensheath or myelinate axons to help restore axonal conduction. Alone, cell transplants are not enough to reestablish substantial function following SCI; however, they could be an essential component of a combination strategy for SCI repair. In the lab, we are working on overcoming some of the limitation of cellular transplants in order to improve their efficacy and facilitate functional recovery.
We are examining and developing strategies to target the cell death that occurs to a substantial number of transplanted cells immediately following transplantation before surviving cells integrate. Using in vivo imaging (IVIS), we are able to visualize the decrease in cells over the first two days following transplantation with bioluminescence reporters and use the IVIS to screen prosurvival strategies. Using ex vivo imaging of spinal cords with the IVIS, we are able to use GFP fluorescence and quantify transplant survival without further tissue processing. Changes in survival detected with the IVIS are then confirmed using microscopy and non-biased stereology or by using biochemistry via Western blotting.
We are also developing and testing approaches to alter the interactions between transplanted cells and host spinal cord cells. Using in vitro co-culture assays, the interactions between cells are being examined. Currently, we are focusing on Schwann cell–astrocyte interactions and how to improve the interaction of the cells at the interface. Both the orientation and the integration of the cells at the edges of the transplant can negatively affect the ability of axons to grow into and out of the transplants. These experiments involve cloning, generation of viral vectors, in vitro co-culture assays, in vivo spinal cord injury modeling, immunohistochemistry, epifluorescence and confocal microscopy, axonal tract tracing and behavioral testing.
Following injury, most axons fail to regrow and connect to their targets. Instead, damaged axons form large, swollen endings that persist for months to years at the lesion margin. These altered endings may represent different types of endings formed in response to different inhibitory factors following injury or a common final phenotype of damaged axons. Surprisingly, little is known about the dystrophic endings that form following SCI. In the lab, we are working on identifying the subcellular changes that occur in axonal endings in vitro in response to inhibitory gradients. We have several lines of research in the lab to identify factors involved in dystrophic axonal ending formation and persistence in an effort to develop novel targets for chronically injured axons.
One of the challenges with targeting dystrophic axonal endings for SCI repair is the limited information currently available regarding the changes that occur within the endings. One of our goals is to establish a means to identify the pool of proteins that are altered within the endings in order to identify novel targets for SCI repair. These studies involve SCI modeling, tract tracing, laser capture microdissection, RNA sequencing and mass spectrophotometry.
To identify potential therapeutic interventions, we are also currently screening pharmacological agents for their ability to increase axonal crossing of proteoglycan gradients using a modification of the spot assay originally developed by Dr. Jerry Silver’s lab. We are interested in further modifying this assay in order to establish a method for generating substrate bound gradients that are compatible for use with microfluidics. This will facilitate the production of more endings for analysis. In addition to establishing methods to enhance the spot assay, we are using immunohistochemistry to identify and screen changes in major subcellular constituents to detect which subcellular factors differ between growth cones and dystrophic axonal endings. Together, these projects involve in vitro experiments to generate dorsal root ganglion neuron spot assay cultures, axonal tract tracing and SCI modeling, and the assessment of axonal endings using immunohistochemistry and epifluorescence and confocal microscopy.
The Hill lab is dedicated to the development and preclinical testing of strategies to enhance regeneration, repair and recovery of function following spinal cord injury. It is our mission that the laboratory be known for doing high quality, reproducible spinal cord injury studies with accuracy and integrity in an innovative and collaborative environment.