Identification Of Regeneration-associated Gene Expression Changes In Composite Regenerative Peripheral Nerve Interfaces (c-rpnis) Reveals Differential Motor And Sensory Phenotypes
Carrie A. Kubiak, MD, Daniel C. Ursu, PhD, Maninder Singh, Paul S. Cederna, MD, Stephen WP Kemp, PhD.
The University of Michigan, Ann Arbor, MI, USA.
PURPOSE: Critical to the design of an ideal bioprosthetic device is the development of a single interface between human and machine that allows for transmission of both afferent somatosensory information and efferent motor signals for device control. The Composite Regenerative Peripheral Nerve Interface (C-RPNI) is a novel biologic interface that demonstrates promise in this role. The C-RPNI is a surgical construct composed of a transected, mixed peripheral nerve implanted between a composite free graft consisting of skeletal muscle and de-epithelialized glaborous skin. We have previously shown that C-RPNI constructs remain healthy and viable for at least 9 months, and are capable of producing both afferent and efferent electrophysiological activity. Previous research has shown that motor and sensory Schwann cells (SC) express different phenotypes which regulate axonal regeneration. In the current study, we performed in-depth molecular analysis to assess potential differences in factors such as cytokines, regeneration associated genes (RAGs), and growth factors between the motor and sensory components of C-RPNIs.
METHODS: 16 rats underwent surgical creation of C-RPNI constructs, and were allowed to convalesce for 3 months. At study endpoint, all C-RPNI constructs were harvested and separated into separate muscle and skin components for genetic analysis. DNA Microarray, Competitive reverse transcriptase-PCR (RT-PCR), 10x Genomics, and RNAseq bioinformatics analysis was performed. A total of ~500 million reads were generated from the 10X Genomics sequencing analysis for each of the C-RPNI replicates. Sequencing data was processed with Cell Ranger software, with further downstream analysis performed using the Seurat R package. All cells with less than 500 genes per cell and more than 25% mitochondrial read content were excluded. RESULTS: Substantial differences were found for both up and down regulated factors between the motor and sensory C-RPNI components. Briefly, mRNA for factors like nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), Oct6, and vascular endothelial growth factor (VEGF) were expressed vigorously by the dermal sensory component. In contrast, mRNA for pleiotrophin (PTN), glial cell-line derived neurotrophic factor (GDNF), c-maf, and connexin-43 was upregulated to a greater degree in the C-RPNI motor component, demonstrating differential motor and sensory phenotypes.CONCLUSION: These findings suggest that both the muscle and dermal components of C-RPNIs upregulate factors in a manner similar to motor and sensory axons during axonal regeneration following nerve injury. These results provide additional support that C-RPNI constructs are appropriate surgical interventions to establish closed loop efferent motor and afferent sensory neural control of advanced prosthetic limbs.
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