Traumatic cervical spinal cord injury (SCI) results in a wide range of outcomes from partial paralysis to complete tetraplegia depending on the location of injury along the length of the cervical spinal cord. Importantly, there is a high density of motor neurons in the cervical region which are involved in important motor outputs such as breathing and hand function. The present study aims to minimize the secondary damage to the spinal cord after the primary insult, by addressing two substantial contributors to neuron death: first, surgical decompression is conducted to reduce local tissue swelling after injury, and second, administration of the metabolite oxaloacetate (OAA) to minimize excitotoxicity by stimulating glutamate transport away from injured neurons. We hypothesized that animals treated with decompression, OAA, or both, would have increased neuronal survival, general tissue sparing, and improved behavioral outcomes than those without treatment, and that combined treatment would be more effective than each individual treatment. We tested the treatments using a clinically relevant rat model for bilateral, moderately severe cervical spinal cord injury. Following treatments, we determined effectiveness by assessing animals’ forelimb function and quantifying motor neuron and white matter sparing in the injured tissue. Results consistent with the hypothesis would have meaningful impacts for future cervical SCI patients, as even a limited increase in tissue sparing in the cervical region have profound functional outcomes for patients’ independence and opportunities. Future studies will work to visualize parameters for segmental tissue at risk after acute injury in order to specify each patient’s treatment and maximize their opportunities for recovery.

Traumatic spinal cord injury (tSCI) often leads to a debilitating loss of sensory, motor, and autonomic function. Currently there are no treatment options available for patients with tSCI. Immediately following the initial trauma, microvessels in the spinal cord rupture, leading to hemorrhage within the spinal cord. Bleeding is a major contributor to a cascade of subsequent injuries, defined as secondary injury, such as swelling, inflammation, and oxidative stress, which results in the expansion of the initial injury. We hypothesize that enhancing blood clotting would limit secondary injury, and subsequently lead to better functional outcomes. To test this, we employed newly developed hemostatic nanoparticles (hNPs), which are designed to localize to the injury site and reduce bleeding in a contusion tSCI model in rodents. The hNPs or control nanoparticles were introduced intravenously within 3 minutes after the injury, and tomato lectin was injected at the end of the experiment to label all patent blood vessels. Clusters of hNPs were found within areas of hemorrhage and blood clot within the injury epicenter, and never seen co-labeled with tomato lectin, suggesting that hNPs were only within parenchyma in areas of active bleeding. Our unique ultrafast contrast enhanced ultrasound (CEUS) imaging was used to visualize hematoma size, local spinal blood perfusion and swelling in real-time. CEUS imaging data showed there was 50% reduction in hematoma size in hNPs treated animals compared to control. We also found significant reductions in hypoperfused volume (50%, p<0.05) as well as spinal cord swelling (30%, p<0.01) in hNPs treated animals compared to controls. Current studies are underway to 1) analyze real-time hemodynamic data obtained from ultrafast CEUS imaging, 2) evaluate chronic 3D blood flow imaging, and 3) quantify functional and histological outcomes from hNP treatment after tSCI.

Unlike humans, zebrafish and salamanders can regenerate entire bony appendages following amputation. This unique regenerative potential may reflect key differences in osteoprogenitor activation and regulation. Here, we used cross-species transcriptomics and single-cell combinatorial indexing RNA sequencing (sci-RNA seq) to 1) identify genes dissimilarly expressed during rat and zebrafish bone regeneration, and 2) determine which cell populations in zebrafish expressed these genes. We modified a previously developed bioinformatics pipeline to include dynamic time warping (DTW) and a robust similarity metric (DTW distance) to quantify gene expression similarity. We analyzed publicly available microarray datasets for zebrafish caudal fin regeneration and rat marrow ablation-induced bone regeneration to determine dissimilarly expressed genes during regeneration. The 30 most similarly expressed genes were enriched with genes of known skeletal function. Of the 30 most dissimilarly expressed genes, 11 were upregulated in zebrafish while downregulated in rat. The most dissimilarly expressed gene during bone regeneration in rat and zebrafish was f13a1b. We performed sci-RNA seq of the regenerating zebrafish caudal fin to investigate these genes at single-cell resolution, identifying what cell types are present in the regenerating fin and how their gene expression varies. f13a1b is expressed within osteoblasts, epidermal, and mesenchymal cells in the regenerating fin. Within the osteoblasts, further clustering identified cycling and non-cycling osteoblasts, pre-osteoblasts (col10a1+), and early-osteoblasts (sp7+). f13a1b expression is enriched in two of the osteoprogenitor populations and less abundant in the later stages of osteoblast differentiation (pre-osteoblasts and early osteoblasts). In conclusion, we have performed comparative transcriptomics to elucidate genes that are upregulated in zebrafish relative to rats and determined the involvement of these genes at single-cell resolution in the regenerating zebrafish caudal fin. Genes upregulated during zebrafish bone regeneration may provide drug targets to improve bone regeneration in humans and a better understanding of the molecular pathways driving appendage regeneration.

Experimental evolution can determine genetic interactions during natural selection in complex systems. Using whole-genome sequencing of 95 parallel populations of haploid Saccharomyces cerevisiae experimentally evolved for 250 generations, we discovered a possible epistatic interaction between two sets of beneficial mutations. The first mutation is a transposable element (TE) insertion into the promoter of FLO1, giving rise to a cellular aggregation phenotype known as flocculation. The second set are putative loss of function mutations in genes encoding members of the SAGA-complex, which is thought to increase expression of genes proximal to TEs. We hypothesize that without the members of the SAGA-complex, the FLO1 gene will be unexpressed, abrogating the flocculation phenotype. We isolated three flocculant clones with TE insertions from different experimental populations and crossed them with three clones with the deletion in the SAGA-complex. Through meiosis, the yeast sporulated into four cells. The ratios of flocculant to wildtype haploid cells are used to determine an epistatic interaction. A 2:2 ratio suggests a non-epistatic interaction while a 1:3 flocculant to wildtype ratio suggests an epistatic interaction. The project is in the early stages but segregation ratios suggest the members of the SAGA-complex with deletion mutations do not hinder the expression of the FLO1 gene. Our alternate hypothesis is members of the SAGA-complex have no effect on the activation of TE insertions that promote the expression of the FLO1 gene. While the initial hypothesis might not hold, this experiment will give us a further understanding of genetic interactions during evolution.