Exogenous Leukemia Inhibitory Factor Stimulates Oligodendrocyte Progenitor Cell Proliferation and Enhances Hippocampal Remyelination

New CNS neurons and glia are generated throughout adulthood from endogenous neural stem and progenitor cells. These progenitors can respond to injury, but their ability to proliferate, migrate, differentiate, and survive is usually insufficient to replace lost cells and restore normal function. Potentiating the progenitor response with exogenous factors is an attractive strategy for the treatment of nervous system injuries and neurodegenerative and demyelinating disorders. Previously, we reported that delivery of leukemia inhibitory factor (LIF) to the CNS stimulates the self-renewal of neural stem cells and the proliferation of parenchymal glial progenitors. Here we identify these parenchymal glia as oligodendrocyte (OL) progenitor cells (OPCs) and show that LIF delivery stimulates their proliferation through the activation of gp130 receptor signaling within these cells. Importantly, this effect of LIF on OPC proliferation can be harnessed to enhance the generation of OLs that express myelin proteins and reform nodes of Ranvier in the context of chronic demyelination in the adult mouse hippocampus. Our findings, considered together with the known beneficial effects of LIF on OL and neuron survival, suggest that LIF has both reparative and protective activities that make it a promising potential therapy for CNS demyelinating disorders and injuries

Impact of Incorporating Movement and Hands-On Experiences on Student Learning

This inquiry took place during my student teaching experience. While student teaching, I explored the use of movement and hands-on experiences throughout the curriculum as a way to effectively engage second graders in learning. I designed and taught lessons incorporating movement, utilized movement breaks, and added movement to classroom transitions in order to answer my research questions of: how can I effectively incorporate movement and hands-on learning into lessons and daily routines; and what impact will incorporating movement and hands-on experiences into the classroom have on student learning? I conclude that movement positively impacts student learning academically, physically, socially, and emotionally. I discovered that the most successful use of movement in the classroom is the natural integration of movement into content area lessons. Movement helps meet the needs of diverse students, increases daily activity for children, and fosters excitement for learning

Dual shell reactor vessel: A pressure-balanced system for high pressure and temperature reactions

The main purpose of this work was to demonstrate the Dual Shell Pressure Balanced Vessel (DSPBV) as a safe and economical reactor for the hydrothermal water oxidation of hazardous wastes. Experimental tests proved that the pressure balancing piston and the leak detection concept designed for this project will work. The DSPBV was sized to process 10 gal/hr of hazardous waste at up to 399{degree}C (750{degree}F) and 5000 psia (34.5 MPa) with a residence time of 10 min. The first prototype reactor is a certified ASME pressure vessel. It was purchased by Innotek Corporation (licensee) and shipped to Pacific Northwest Laboratory for testing. Supporting equipment and instrumentation were, to a large extent, transported here from Battelle Columbus Division. A special air feed system and liquid pump were purchased to complete the package. The entire integrated demonstration system was assembled at PNL. During the activities conducted for this report, the leak detector design was tested on bench top equipment. Response to low levels of water in oil was considered adequate to ensure safety of the pressure vessel. Shakedown tests with water only were completed to prove the system could operate at 350{degree}C at pressures up to 3300 psia. Two demonstration tests with industrial waste streams were conducted, which showed that the DSPBV could be used for hydrothermal oxidation. In the first test with a metal plating waste, chemical oxygen demand, total organic carbon, and cyanide concentrations were reduced over 90%. In the second test with a munitions waste, the organics were reduced over 90% using H{sub 2}O{sub 2} as the oxidant

Use of high-content imaging to quantify transduction of AAV-PHP viruses in the brain following systemic delivery

The engineering of the AAV-PHP capsids was an important development for CNS research and the modulation of gene expression in the brain. They cross the blood brain barrier and transduce brain cells after intravenous systemic delivery, a property dependent on the genotype of Ly6a, the AAV-PHP capsid receptor. It is important to determine the transduction efficiency of a given viral preparation, as well as the comparative tropism for different brain cells; however, manual estimation of adeno-associated viral transduction efficiencies can be biased and time consuming. Therefore, we have used the Opera Phenix high-content screening system, equipped with the Harmony processing and analysis software, to reduce bias and develop an automated approach to determining transduction efficiency in the mouse brain. We used R Studio and ‘gatepoints’ to segment the data captured from coronal brain sections into brain regions of interest. C57BL/6J and CBA/Ca mice were injected with an AAV-PHP.B virus containing a green fluorescent protein reporter with a nuclear localization signal. Coronal sections at 600 μm intervals throughout the entire brain were stained with Hoechst dye, combined with immunofluorescence to NeuN and green fluorescent protein to identify all cell nuclei, neurons and transduced cells, respectively. Automated data analysis was applied to give an estimate of neuronal percentages and transduction efficiencies throughout the entire brain as well as for the cortex, striatum and hippocampus. The data from each coronal section from a given mouse were highly comparable. The percentage of neurons in the C57BL/6J and CBA/Ca brains was approximately 40% and this was higher in the cortex than striatum and hippocampus. The systemic injection of AAV-PHP.B resulted in similar transduction rates across the entire brain for C57BL/6J mice. Approximately 10–15% of all cells were transduced, with neuronal transduction efficiencies ranging from 5% to 15%, estimates that were similar across brain regions, and were in contrast to the much more localized transduction efficiencies achieved through intracerebral injection. We confirmed that the delivery of the AAV-PHP.B viruses to the brain from the vasculature resulted in widespread transduction. Our methodology allows the rapid comparison of transduction rates between brain regions producing comparable data to more time-consuming approaches. The methodology developed here can be applied to the automated quantification of any parameter of interest that can be captured as a fluorescent signal

Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing

Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies