Analysis Of Differential Gene Expression In Chronically Denervated Nerve Tissue Using RNA Sequencing
Kimberly H. Khoo1,Alison L. Wong1,Thomas Harris1, Connor Glass1,Nicholas von Guionneau1,Erica Lee1,Alex Serafini2,Nicholas Hricz1,Harsha Malapati1,Ruifa Mi1,Vivek Swarup3,Riki Kawaguchi1,Ahmet Hoke1,Sami Tuffaha1
1Department of Plastic and Reconstructive Surgery, Johns Hopkins University, Baltimore, MD 2Icahn School of Medicine at Mount Sinaicahn School of Medicine at Mount Sinai, New York City, 3New York University of California Irvine, Irvine, CA
Background: Denervation atrophy of both muscle and Schwann cells (SC) contribute to poor functional recovery after delay in peripheral nerve repair. Previous work by the authors has demonstrated that periods of chronic denervation greater than 12 weeks resulted in statistically significantly poorer functional recovery. With this understanding, we hoped to discern the changes in RNA expression that may be implicated in this differential functional recovery over time.
Methods: Male, adult Lewis rats underwent unilateral forelimb denervation of the ulnar nerve at 1, 8, 12, 16, and 24 weeks. After the allotted denervation periods, both the median and ulnar nerves were harvested bilaterally. The nerves were homogenized, RNA was purified, and then RNA-sequencing was conducted using the TrueSeq RiboZero gold kit. Data were analyzed and underwent quality control using FastQC, aligned to reference genome Rnor6.1 on STAR, and quantified as Transcripts Per Million (TPM) using Salmon. Principle component analysis was performed on the data, then a linear mixed-effects method was used to perform Differential Gene Expression analysis, controlling for nerves harvested from the same animal. The false-discovery rate was set at <.05, and the absolute beta value >0.1.
Results: A total of 1624 genes were identified as being differentially expressed, of which 327 genes were upregulated and 1297 genes were downregulated. Gene ontology demonstrated that upregulated genes were involved in regulation of immune cells, such as lymphocytes and neutrophils, and downregulated genes were implicated in axonogenesis, axon guidance, and cytoskeleton-dependent intracellular transport. Preliminary network analysis using QIAGEN Ingenuity Pathway Analysis likewise suggested that downregulated inflammatory pathways are central to the differentially expressed genes implicated in chronic nerve denervation.
Conclusion: RNA-seq analysis demonstrated that genes implicated in various inflammatory pathways and cellular mechanisms, such as cytoskeletal transport and axonal guidance, were differentially expressed with varying durations of peripheral nerve denervation. Further analysis using established gene ontology databases and enrichment tools are needed to elucidate which pathways are most impactful in peripheral nerve injuries and how best to tailor interventions to promote recovery.
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