Photomechanical Wave-Driven Delivery of siRNAs Targeting Intermediate Filament Proteins Promotes Functional Recovery after Spinal Cord Injury in Rats

Takahiro Ando,Hiroaki Kobayashi,Minoru Obara,Terushige Toyooka,Hiroshi Nawashiro,Hiroshi Ashida,Shunichi Sato,Shu-Min Duan

doi:10.1371/journal.pone.0051744

Takahiro Ando, Hiroaki Kobayashi + Show 6 more

Open Access

https://doi.org/10.1371/journal.pone.0051744

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Journal: PloS one	Publication Date: Dec 14, 2012
Citations: 93	License type: CC BY 4.0

Affiliation: National Defense Medical College, Keio University

Abstract

The formation of glial scars after spinal cord injury (SCI) is one of the factors inhibiting axonal regeneration. Glial scars are mainly composed of reactive astrocytes overexpressing intermediate filament (IF) proteins such as glial fibrillary acidic protein (GFAP) and vimentin. In the current study, we delivered small interfering RNAs (siRNAs) targeting these IF proteins to SCI model rats using photomechanical waves (PMWs), and examined the restoration of motor function in the rats. PMWs are generated by irradiating a light-absorbing material with 532-nm nanosecond laser pulses from a Q-switched Nd:YAG laser. PMWs can site-selectively increase the permeability of the cell membrane for molecular delivery. Rat spinal cord was injured using a weight-drop device and the siRNA(s) solutions were intrathecally injected into the vicinity of the exposed SCI, to which PMWs were applied. We first confirmed the substantial uptake of fluorescence-labeled siRNA by deep glial cells; then we delivered siRNAs targeting GFAP and vimentin into the lesion. The treatment led to a significant improvement in locomotive function from five days post-injury in rats that underwent PMW-mediated siRNA delivery. This was attributable to the moderate silencing of the IF proteins and the subsequent decrease in the cavity area in the injured spinal tissue.

Highlights

The absence of spontaneous axonal regeneration after spinal cord injury (SCI) is attributed to the lack of neurotrophic factor support [1,2,3] and to the presence of extracellular matrix inhibitors, such as chondroitin sulfate proteoglycans and myelin-associated inhibitors [4,5,6,7], and glial scar formation around the injury site [8,9,10]
Characteristics of photomechanical waves (PMWs) used for Gene Transfection To evaluate the propagation characteristics of PMWs through the spinal cord, temporal pressure profiles of PMWs before and after propagation through an extracted spinal cord were measured using a hydrophone under the conditions used for small interfering RNAs (siRNAs) transfection (Fig. 1A)
The proliferation of reactive astrocytes is accompanied by an increase in the production of intermediate filament (IF) proteins such as glial fibrillary acidic protein (GFAP) and vimentin [18,19,20,58,59]

Summary

Introduction

The absence of spontaneous axonal regeneration after spinal cord injury (SCI) is attributed to the lack of neurotrophic factor support [1,2,3] and to the presence of extracellular matrix inhibitors, such as chondroitin sulfate proteoglycans and myelin-associated inhibitors [4,5,6,7], and glial scar formation around the injury site [8,9,10]. One of the main molecular hallmarks of reactive astrocytes is the up-regulation of intermediate filament (IF) proteins such as glial fibrillary acidic protein (GFAP) and vimentin [17,18,19]. Overexpression of these IF proteins causes glial scar formation, resulting in a physical and biochemical barrier for axonal regeneration after SCI. Double knockout mice lacking GFAP and vimentin showed lower levels of astroglial activity and astrocytic hypertrophy, and exhibited reduced scar formation after spinal cord hemisection [20]

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Conclusion