inducing freezing injury to establish an orofacial muscle fibrosis mouse model

Stabilire un modello di fibrosi muscolare orofacciale nei topi tramite una lesione da congelamento

Orofacial muscles are critical in daily physiological activities such as mastication, speech, respiration, and facial expression1. In congenital orofacial deformities, however, these muscles exhibit atrophic and fibrotic alterations, leading to impaired body health and social cognition2. Facial reconstructive surgery remains the first-line treatment, but up to 30-70% of postoperative patients still suffer from muscle loss and muscle dysfunction3,4 The failure of orofacial muscle regeneration has been attributed to intrinsic factors, which cannot be corrected by surgery alone. 
The emergence of orofacial muscles is an evolutionary novelty, accompanying the complex vertebrate head and chambered heart5,6. Unlike their somite-derived limb counterparts, orofacial muscles originate from the branchial arch7. These phylogenetic and ontogenetical characters may predispose them to distinct regenerative behaviors8. It has been reported that the masseter (MAS) muscle developed severe fibrosis at the time when the tibialis anterior (TA) muscle fully regenerated after exposure to the same extent of injury1,9. However, the underlying mechanism of regeneration remains poorly understood. 
In this study, a freezing injury model of the mice masseter muscle was established to facilitate the investigation into orofacial muscle regeneration. We chose 14 days after injury as the time point for assessing fibrosis phenotype as it was the earliest time point where discernible divergence was detectable between two muscles. Complete regeneration of the MAS after injury requires at least 40 weeks1. Consistently, this study revealed a remarkable deposition of collagen following freezing injury of MAS compared to the regular regeneration of the TA at 14 days post injury. With the help of this model, further mechanistic studies of muscle atrophy and fibrosis can be carried out, which will in turn help the development of potential therapeutic avenues to promote orofacial muscle regeneration after surgery.

Autore in primo piano: Esplorare la rigenerazione dei muscoli orofacciali - Approfondimenti e innovazioni

Lesione da congelamento nel muscolo massetere del topo per stabilire un modello di fibrosi muscolare orofacciale

Current skeletal muscle regeneration studies merely focus on the limb and the trunk muscle, leaving the characteristics and mechanism of orofacial muscle regeneration largely unexplored. The scope of this research is to create and easier and reliable mouse model to facilitate further investigation into muscle regeneration in the orofacial area. Recent developments review that orofacial muscles constitute a unique subset of skeletal muscle with a distinct evolutionary trajectory and developmental origin.They also exhibit different regeneration performances compared with their limb counterparts. A deeper investigation into the orofacial muscle regeneration is warranted to figure out the mechanism underlying these marked differences. The study present a rapid and reproducible method to study orofacial muscle regeneration.The half-freezing injury process took 20 minutes, and the harvest process took only 10 minutes for each mouse, and the mice can tolerate the injury well without any impairment of locomotion. Since our model allows for subsequent comparison between the fibrotic masseter muscle and the fully regenerated tibialis anterior muscle from the aspects of morphology, histology, and molecular regulation, it serves us a power for two for us to delve deeper into the underlying mechanism. Our future research will focus on the causal effects among the alterations of the resident muscle stem cells and multiple niche cells, and correspondingly developed interventions to the process of orofacial muscle fibrosis.

Indurre una lesione da congelamento per stabilire un modello murino di fibrosi muscolare orofacciale

Per iniziare, posizionare il mouse anestetizzato sul tavolo operatorio. Utilizzando un batuffolo di cotone, applicare la pasta depilatoria sulla pelle, coprendo il muscolo massetere e il muscolo tibiale anteriore. Dopo aver atteso uno o due minuti, lavare via la pasta con una salvietta chirurgica.Per esporre il muscolo massetere, palpare prima lungo il bordo inferiore della mandibola. A cinque millimetri dal bordo, tagliare la pelle lungo la linea, collegando la commessura orale al trago. Per eseguire il congelamento, utilizzare una pinza pre-raffreddata per trattenere un pezzo di ghiaccio secco.Posiziona il ghiaccio secco lungo gli assi lunghi dei muscoli masseteri direttamente sulla superficie del muscolo e mantieni la posizione per cinque secondi. Verifica che il muscolo appaia rigido e bianco pallido subito dopo aver rimosso il ghiaccio secco, quindi ritorna al suo colore e consistenza normali entro 22-25 secondi. Dopo che il muscolo si è completamente ripreso, chiudere la ferita cutanea con almeno tre punti di sutura 7-0.Per il muscolo tibiale anteriore, palpare lungo il polpaccio per individuare il bordo anteriore dell'osso tibiale. Due millimetri più avanti rispetto al bordo, tagliare la pelle, partendo dal ginocchio e terminando alla caviglia. Per eseguire il congelamento, utilizzare una pinza pre-raffreddata per trattenere un pezzo di ghiaccio secco.Posizionare il ghiaccio secco lungo gli assi lunghi dei muscoli tibiale anteriori direttamente sulla superficie e tenere premuto per cinque secondi. Verifica che il muscolo appaia rigido e bianco pallido subito dopo aver rimosso il ghiaccio secco, quindi ritorna al suo colore e consistenza normali entro 22-25 secondi. Dopo aver suturato la ferita cutanea, posizionare il mouse su una piastra riscaldante mantenuta a circa 38 gradi Celsius.Monitorare continuamente fino a quando il mouse non recupera il suo riflesso raddrizzante.

inducing-freezing-injury-to-establish-an-orofacial-muscle-fibrosis

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JoVE Journal

Biology

Inducing Freezing Injury to Establish an Orofacial Muscle Fibrosis Mouse Model

Freezing Injury in Mouse Masseter Muscle to Establish an Orofacial Muscle Fibrosis Model

Orofacial muscle constitutes a subset of skeletal muscle tissue, with a distinct evolutionary trajectory and development origin. Unlike the somite-derived ...