Biophysical stimulation induces demyelination via an integrin‐dependent mechanism

Abstract

Objective: Chronic nerve compression (CNC) injuries occur when peripheral nerves are subjected to sustained mechanical forces, with increasing evidence implicating Schwann cells as key mediators. Integrins, a family of transmembrane adhesion molecules that are capable of intracellular signaling, have been implicated in a variety of biological processes such as myelination and nerve regeneration. In this study, we seek to define the physical stimuli mediating demyelination and to determine whether integrin plays a role in the demyelinating response. Methods: We used a previously described in vitro model of CNC injury where myelinating neuron-Schwann cell cocultures were subjected to independent manipulations of hydrostatic pressure, hypoxia, and glucose deprivation in a custom bioreactor. We assessed whether demyelination increased in response to applied manipulation and determined whether integrin-associated signaling cascades are upregulated. Results: Biophysical stimulation of neural tissue induced demyelination and Schwann cell proliferation without neuronal or glial cytotoxicity or apoptosis. Although glucose deprivation and hypoxia independently had minor effects on myelin stability, together they potentiated the demyelinating effects of hydrostatic compression, and in combination, significantly destabilized myelin. Biophysical stimuli transiently increased phosphorylation of the integrin-associated tyrosine kinase Src within Schwann cells. Silencing this integrin signaling cascade blocked Src activation and prevented pressure-induced demyelination. Colocalization analysis indicated that Src is localized within Schwann cells. Interpretation: These results indicate that myelin is sensitive to CNC injury and support the novel concept that myelinating cocultures respond directly to mechanical loading via activating an integrin signaling cascade.

The relationship of nerve fibre pathology to sensory function in entrapment neuropathy

“…In experimental models, both acute (Ochoa et al , 1971; Dyck et al , 1990) and chronic nerve compression (Mackinnon et al , 1984 b ) interfere with the normal architecture of nodes of Ranvier and their adjacent paranodes at the compression site, presumably by ischaemia (Nukada et al , 1985) and mechanical deformation of Schwann cells (Pham and Gupta, 2009; Lin et al , 2012). Our findings demonstrate however that such changes are not confined to the entrapment site but significantly altered axoglial relationships are confirmed in distal innervation targets.…”

Early Surgical Decompression Restores Neurovascular Blood Flow and Ischemic Parameters in an in Vivo Animal Model of Nerve Compression Injury

“…The primary antibodies used were rabbit anti-HIF1a (1:1000 dilution; Novus Biologicals, Littleton, Colorado), mouse anti-MMP9 (1:5000 dilution; Abcam, Cambridge, Massachusetts), rabbit anti-MMP2 (1:5000 dilution; abcam), rabbit anti-superoxide dismutase (1:5000 dilution; Sigma-Aldrich, St. Louis, Missouri), mouse anti-catalase (1:5000 dilution; Sigma-Aldrich), mouse anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1:50,000 dilution; Fitzgerald, Acton, Massachusetts), and mouse anti-tubulin (1:10,000 dilution, Sigma-Aldrich) 19,[25][26][27][28][29][30][31] . The western blotting procedure was performed at two, four, and six weeks after injury in the no-decompression groups as well as at two and six weeks after treatment in the decompression groups.…”

“…Briefly, 75 to 100 mg of protein was separated by 8% or 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), transferred to nitrocellulose membrane, blocked with 5% dry skimmed milk, and incubated overnight at 4°C with primary antibodies. Donkey anti-mouse secondary antibody conjugated with horseradish peroxidase (HRP, 1:10,000 dilution; EMD Millipore, Billerica, Massachusetts) or goat anti-rabbit secondary antibody conjugated with HRP (1:10,000 dilution) was used for detection 19,25 . Blot development was performed with Immobilon…”

“…The compressed nerve has increased vascular permeability and neural vascularity with an upregulation of vascular endothelial growth factor (VEGF) and its Schwann cell receptors (fetal liver kinase receptor and fms-like tyrosine kinase receptor) at early time points, followed by a dramatic decrease [16][17][18] . Recent data have revealed that hypoxia potentiates the demyelinating effect of hydrostatic compression in an in vitro model of nerve compression injury 19 , but the role that ischemia plays in triggering the pathological responses in such an injury still remains unknown.…”

Reply

“…As human tissue from early compression neuropathy is not currently available secondary to the morbidity that would occur from harvesting neural tissue in electrophysiologically confirmed compression neuropathy, these animal models are meant to provide in vivo data and findings so that we can better explore the mechanisms of this condition with in vitro models of compression neuropathy. [5][6][7] We have used this combined in vivo and in vitro modeling of compression neuropathies for the past decade to hopefully develop meaningful nonoperative treatment strategies in the coming years. 1.…”