When treating neurological diseases, the uptake, penetration, and cellular interaction of a therapeutic within the brain is critical to the success of the therapeutic. However, the means to gather real-time molecular information from the diseased brain is limited, and high-throughput platforms that can assay sub-micron changes in tissue in the presence of injury are still lacking. The Nance lab utilizes nanotechnology to probe the brain microenvironment in the presence of injury. We integrate in vitro to in vivo models with imaging, molecular biology, and data science tools to extract statistically relevant information that captures changes in the brain that might influence how a therapeutic behaves. We have used the information gathered from application of these tools to design nano-based therapeutics that can achieve region and cell-specific targeting in the brain for improved neuroprotection in a variety of brain injury models. Check out some of our active project areas below.
Developing slice culture models of injury processes
There is a significant lack of effective translational models and biological markers of disease states. Models that capture disease processes are not only essential for improving our understanding of neurological mechanisms that underlie disease, but also for providing patient screening tools to increase the probability of success for drug delivery platforms as they enter clinical trials.
Organotypic whole hemisphere (OWH) brain slice models provide a simplified ex vivo system that preserves brain structure and cell function, while capturing all regions of the brain. We have adapted our OWH slice models from organotypic hippocampal (HC) slice cultures, which have been used to study the effects of various toxins, and small molecule agonists or inhibitors. We have created OWH slice models of inflammation, oxidative stress, and glutamate excitotoxicity. We also culture brain slices taken from in vivo injury models, including from rat, ferret, rabbit, and mouse models of neonatal or perinatal brain injury.
Research in this area involves a variety of experimental techniques, from slice preparation and culturing to assays for cell viability, RNA and protein production, and methods for live and fixed cell imaging. Each of these experiments requires methodology development to adapt techniques currently used for in vitro cell culture or in vivo tissue processing.
While our primary focus is on the brain, we also look at applications in the lung and vaginal tract, and are interested in exploring research efforts in the liver and kidney.
Measuring transport in living tissue
We are interested in developing tools that allow us to real-time track and quantify the movement of a variety of entities in living tissue. In particular, we focus on the transport of nanoparticles, the mobility of cells, and the trafficking of extracellular vesicles. We look to characterize transport as a function of physiological factors, age, brain region, and disease.
There are four components to this research area that we focus on: (1) creating viable slice platforms that can adapt to multiple species, organs, and disease models, (2) engineering well-characterized probe nanoparticles with a variety of physicochemical properties, (3) implementing a high spatial resolution setup for real-time and time-lapse imaging of nanoparticles and cells in living tissues, and (4) creating data science and machine learning tools to extract statistically significant information from the acquired imaging data, whether still images or videos.
We often look to directly measure multi-scale changes in ECM microstructure in real-time and over time in living tissue. We use multiple particle tracking (MPT) to measure changes in the “pore” or mesh spacing of extracellular matrices. In the brain, individual nanoparticle behavior is affected by the geometry of the extracellular space and extracellular matrix, and by interactions with the extracellular matrix. We can measure anticipated changes in this microstructure through quantifying an estimated mesh spacing and local viscosity.
When we track cells, we measure the movement of the cell bodies (somas) and cell branches (processes). We can look at an individual cell type or how two cells, like a microglia and neuron, might interact with each other. This allows us to observe some functional changes in the brain.
Engineering nanotherapeutics for neuroprotection in the developing injured brain
When delivery limitations of a nanoparticle platform are better understood, an optimal formulation can be engineered and evaluated for therapeutic efficacy in clinically relevant animal models of brain disease. We focus on developing nanoparticle-based therapeutic approaches for improved neurological outcomes in perinatal/neonatal brain injury, specifically those where neuroinflammation, oxidative stress, and glutamate toxicity play key roles. We are developing nanoparticles that can specifically target regions of injury, and uptake in cells involved in injury. Our current platforms are capable of selectively localizing in either microglia or neurons.
We often develop nanotherapeutic platforms that utilize a drug with a solubility and/or delivery problem - that is, the drug either is not soluble in fluids like blood or water, and it is not able to reach its target site in high enough concentrations to be effective. We also look for drugs that are broad-acting and can affect multiple pathways. We explore small and large molecule drugs, as well as proteins or enzymes. Our therapeutic nanoparticle platforms are polymer-based, and often incorporate materials that have been utilized extensively in adult populations but have not been applied to children or newborns.
We are focused on neonatal or pediatric brain injury models. These include autism, depression, cerebral palsy, hypoxia ischemia, and neurodevelopmental disability. Beyond the models we focus on in our group, we work closely (daily) with clinical collaborators in Neonatology and Neurology. There is currently no effective cure for any of these diseases, therefore we have a lot of opportunity to have impact.
Characterizing animal models of developmental brain disease
In an effort to gain a better understanding of developmental brain disease and injury, we have generated two models of neurodevelopmental disease. One is a transgenic rat model with hallmarks of autism. The other is an inflammation-mediated model of pre-adolescent depression in rats. We utilize imaging analysis (immunohistochemistry, histopathology), behavioral analysis, and RNA and protein analysis (RT-PCR, FACS, western blot) to characterize how the injury develops and impacts normal brain function.
In addition, we collaborate with Dr. Juul, Dr. Wood, and Dr. Garden to study how prematurity disrupts normal brain function, and how brain injury early in life, especially in the preterm infant, might increase risk to injury (TBI, stroke, concussion) later in life. Through these collaborations, we implement our imaging tools to examine how cellular and extracellular changes lead to loss of normal brain function.