Peripheral Nerve Matrix Hydrogel Promotes Recovery by Modulation of Macrophage Phenotype
Jonathan Cheetham, VetMB, PhD, DACV1; Megan Bernard, MS, DVM2; Travis Prest, MS3; Tim P. Moore, BS2; Lorenzo Soletti, PhD, MBA, MS4; Brown Bryan, PhD3
1Department of Clinical Sciences, Cornell University College of Veterinary Medicine, Ithaca, NY, 2Cornell University College of Veterinary Medicine, Ithaca, NY, 3McGowan Institute for Regenerative Medicine, Pittsburgh, PA, 4Renerva LLC, Pittsburgh, PA
Introduction
Macrophages and Schwann cells play a key role in the orchestration of early events after peripheral nerve injury. One opportunity for functional improvement after nerve reconstruction is manipulation of the microenvironment at the site of nerve repair to promote modulation of the host inflammatory response and promote Schwann cell (SC) migration and axon extension across the repair site. We have recently shown that this is an effective approach.
Methods
Here we determined the effects of a decellularized peripheral nerve matrix (PNM) hydrogel on macrophage gene expression, phenotype and migration using a combination of FACS, Nanostring technology and immunohistochemistry. We also evaluate effects on SC migration, numbers of motor neurons reaching their target and axon extension using a combination of mouse and rat models. Finally, functional recovery was determined after both sciatic crush injury and common peroneal nerve transection associated with a short gap. Control groups including uninjured animals, exposure of the nerve without injury, transection and ligation without repair and delivery of hydrogel to uninjured nerves were also performed (n=8/group). Animals were followed for 12 weeks and assessed longitudinally using multiple measures of sensory and functional recovery. Metrics included von Frey nociception assay, sciatic functional index, and kinematic analysis. At 12 weeks animals were subjected to electrophysiologic assessment of evoked compound motor action potential prior to euthanasia for tissue collection and subsequent histologic analysis.
Results
PNM promoted expression of Platelet Derived Growth Factor and M2 (regenerative) genes including Chi313, Retnla and IL4ra. Macrophage, particularly M2 (regenerative phenotype), recruitment to the injury site was increased in both mice and rats (2 fold increase, p<0.05). PMN promoted SC migration both in vitro and in vivo (p<0.05). Axon extension determined by extension of GFP+ axons across an 8mm gap was increased in the presence of PNM compared with an empty conduit (>5 fold increase, p<0.05). PNM increased the number of retrograde labelled MN at 12 weeks after injury compared to empty conduit (20% p<0.05).
The amplitude of Compound Motor Action Potentials across the sciatic crush injury site, demonstrating axonal regrowth to the terminal muscle was increased by 50% compared to control following subepineural delivery of PNM 12 weeks after nerve crush.
Conclusion
These results demonstrate that an injectable, peripheral nerve matrix hydrogel derived from porcine sciatic nerve can modulate the response of two key cell types which conduct the early response to nerve injury.
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