Dataset Information

In vivo injection of α-bungarotoxin to improve the efficiency of motor endplate labeling.

ABSTRACT:

Introduction

Motor endplates are composed of a motor neuron terminal and muscle fiber and are distributed in skeletal muscle, causing muscle contraction. However, traditional motor endplate staining methods are limited to the observation of partial skeletal muscle. The procedure was time-consuming due to strict incubation conditions, and usually provided unsatisfactory results. We explored a novel method to label motor endplate rapidly by in vivo injection of fluorescent α-bungarotoxin.

Methods

Fifty-two mice were randomly divided into two groups, an experiment group (n = 50), and a contrast group (n = 2). In experiment group, α-bungarotoxin was injected via the caudal vein. The injection dosages were designated as 0.1, 0.2, 0.3, 0.4, and 0.5 μg/g. The experimental mice were divided into five subgroups of ten mice per group. The contrast group was only injected with 200 μL normal saline solution. Bilateral gastrocnemius were acquired for microscope analysis and optical clearing to seek specific fluorescent signal.

Results

A dose of 0.3 μg/g of α-bungarotoxin with 1 h conjugation time could display the number and structure of motor endplate in plane view. Compared with the traditional procedure, this method was rapid, convenient, and time-saving. Combined with the optical clearing technique, spatial distribution could also be seen, helping to better understand the stereoscopic view of motor endplate position in skeletal muscle.

Conclusions

In vivo injection of α-bungarotoxin proved effective for studying motor endplate in skeletal muscle.

SUBMITTER: Chen W

PROVIDER: S-EPMC4851864 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectiveTissue engineering of the larynx requires a complex, multiple tissue layer design. Additionally, spontaneous reinnervation of the larynx after recurrent laryngeal nerve (RLN) injury is often disorganized, resulting in subpar function. This study investigates use of tissue-engineered cartilage and motor endplate-expressing (MEE) tissue-engineered skeletal muscle implants for laryngeal reconstruction and the promotion of organized reinnervation after RLN injury.MethodsF344 rat primary muscle progenitor cells (MPCs) were isolated. Three-dimensional muscle constructs were created by encapsulating MPCs in type I oligomeric collagen under passive tension. Constructs were then cultured in differentiation medium (MPC control constructs) or induced to form motor endplates (MEE constructs) with neurotrophic agents. Three-dimensional cartilage constructs were created with adipose stem cells differentiated in chondrocyte medium. The muscle and cartilage constructs were implanted into surgically created myochondral defects in the F344 rat larynx with injured or intact (control) RLN. At 1-, 3-, and 6-month timepoints, videolaryngoscopy, electromyography (EMG), histology, and immunohistochemistry were used to assess outcomes.ResultsAt all timepoints, cartilage-muscle implants were well integrated into host tissue. Functionally, there was increased vocal fold adduction and EMG activity in nerve-injured rats treated with the MEE constructs when compared to those treated with the MPC control constructs. Motor endplate-expressing constructs had increased myofiber cross-sectional area compared to MPC control constructs.ConclusionAlthough our laboratory previously demonstrated that muscle and cartilage constructs could be used separately for hemilaryngeal reconstruction, this study suggests combining them with the modification of MEEs rather than MPCs, resulting in improved muscle recovery after recurrent laryngeal nerve injury.Level of evidenceNA Laryngoscope, 129:1293-1300, 2019.