Virginia Research Day 2025

Medical Student Research Biomedical

12 RNA Seq Profile and Gene Ontology of the Acute Phase of a Mild Spinal Cord Injury

Krystalee P. Revanna 1 ; Emmaline Lang 1 ; Carly Norris 2 ; Susan Murphy 2,3 ; Pamela VandeVord 2,3,4 ; Ramu Anandakrishnan 1,5 ; Kelly C.S. Roballo 1,5 ; Kelly Roballo, DVM, PhD Corresponding author: krevanna@vcom.edu 1 Edward Via College of Osteopathic Medicine - Virginia Campus 2 School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, VA, United States 3 Department of Biomedical Engineering and Mechanics, Virginia Tech, Blacksburg, VA, United States 4 Veterans Affairs Medical Center, Salem, VA, United States 5 Department of Biomedical Sciences and Pathobiology, Virginia Maryland College of Veterinary Medicine

Previous work from the VandeVord group developed a non-invasive blast model of spinal cord injury (SCI) using rats. However, the characterization of the model was limited to acute phases. However, it was noted that there was no resulting paralysis, leading to the hypothesis that this model is inducing more of a sensory/pain injury. In this project, we used the blast SCI (bSCI) animal model to evaluate RNA expression post-injury. The model uses the advanced blast simulator located within the VT Center for Injury Biomechanics to generate a closed, non surgical injury, resulting from a blast trauma5. Our preliminary data led us to transition the model to ‘spinal cord contusions’ with resulting pain behaviors. Clinical reports observe that after a SCI from impact trauma, individuals experience motor dysfunction, paralysis, and pain. It is known that inflammatory processes are initiated in the first 72 hours post-injury which leads to tissue damage, loss of function, and pain response6. In this project, the bSCI model was utilized to examine, level of injury and tissue response

after bSCI at the RNA level. For that, we used male rats with 250-300g with 8 animals per group which included a sham group, no injured animals, and injured, animals that have experienced blunt, isolated, spinal cord trauma. All procedures performed in this project received IACUC approval (# 22-182) and followed all laboratory animal guidelines and ethical procedures. At 24 hrs or 72 hrs post injury, animals were euthanized following all the animal guidelines, and spinal cords were isolated for RNA isolation. RNA sequence was done following the next steps. First freshly isolated bSCI or Sham spinal cord cells were resuspended in 100 ml of PicoPure Extraction Buffer (Thermo Fisher Scientific), and then stored at -80C. RNA isolation will be performed using the PicoPure RNA isolation kit (Thermo Fisher Scientific), and samples with high quality RNA (RNA integrity number, .7.0) as measured using the 4200 TapeStation (Agilent Technologies) were used for library preparation. The next steps,

library preparation and sequence, were performed at Genomics Sequencing Center- Fralin Life Sciences VT, and was followed by demultiplexing, alignment and normalization. For RNA seq analysis, we used an approach that includes setting low count filtering, establishing a noise threshold, checking for potential outliers, running appropriate statistical tests to identify differentially expressed genes (DEGs), clustering of genes by expression pattern, and testing for gene ontology (GO) enrichment. GOrilla was utilized to analyze the genes that showed significant changes in regulation at 24 and 27 hours post injury. The purpose of this was to determine the link between the genes. Results revealed that amongst the genes that were upregulated and down regulated post injury, the commonalty was nucleic acid binding. This implies that nucleic acid biding is involved in the post inflammatory response. With this, better management and treatments can be implemented.

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2025 Research Recognition Day