Duchenne muscular dystrophy (DMD) has been described as one of the most devastating, rapidly progressive and severe forms of hereditary myopathies, with an incidence of 1 in 3,500 males born [1, 2]. DMD is an X-linked recessive disease caused by a mutation in the gene encoding a 427-kDa protein located on the short arm of chromosome X at locus Xp21 .
Dystrophin represents only 0.002% of the striated muscle cell mass and is located at the intracellular surface of the sarcolemma in combination with several integral membrane glycoproteins, forming the dystrophin-associated glycoprotein complex (DGC). The DGC is responsible for the membrane permeability of muscle cells that promotes the binding of F-actin, a thin myofilament protein, to dystroglycan, forming the DGC of cardiac and skeletal striated muscle cells and smooth muscle cells . Although the function of the DGC has not been elucidated in the literature, it may have a role in cytoskeleton structural integrity and cell survival signalling .
The first symptoms of DMD are usually reported at 2 years of age, but parents only recognise them at approximately age 5, when the child begins to have difficulties going up or down stairs and falls . At approximately 20 to 30 years of age, depending on the ventilation resources used, the patients die as a result of respiratory muscle impairment, which is the cause of death in 80% of DMD patients, due to respiratory failure in addition to an infection or heart failure. Structural deformities are characteristic of DMD patients, resulting from their confinement to a wheelchair. The most serious structural deformity is scoliosis, the main culprit underlying the drastic reduction in lung function. Due weakening of respiratory muscle, these patients are unable to generate respiratory pressure (maximal inspiratory pressure and maximal expiratory pressure), and the peak expiratory flow is reduced. Thus, all of these factors lead to the premature death of DMD patients [3, 7, 8]. The main failure of muscles involved in breathing occurs in the diaphragm. The reduction in lung compliance and chest wall mobility leads to an increased mechanical load sustained with each breath. The onset of hypercapnia, pulmonary hypoventilation, hypoxaemia (following hypercapnia) and, consequently, the clearance of secretions results from weakened musculature. The uses of mechanical forces acting on the body or intermittent pressures acting on the airways have helped to improve the performance of inspiratory and expiratory muscles. Among them, negative pressure body ventilators (NPBVs) and bilevel positive airway pressure (BiPAP) improve the quality of life of patients but do not prevent the progression of respiratory failure .
Similarly to human DMD, in mdx mice (X chromosome-linked muscular dystrophy in the mouse) muscle inflammation begins at 3 weeks of age, peaking between 8 and 12 weeks. After this period, inflammation disappears spontaneously from the muscles. However, the mdx diaphragm muscle shows moderate endomysial fibrosis that is aggravated by intense oxidative stress at 3 months of age and worsens at 6 months of age without the clearance of inflammation . This disease progression is similar to human DMD [10, 11].
The life span of the mdx mouse is reduced, and respiratory and/or cardiac insufficiency is the main cause of death [12, 13]. Recent studies performed using whole-body plethysmography have shown that the respiratory rate, tidal volume and minute volume are significantly reduced in the mdx animals from 2 to 6 months of age and may worsen at 7 months of age [13, 14]. In contrast, some less recent data report that there is only a small difference in the tidal volume (Vt) in response to hypercapnia in mdx mice at 7 months and little ventilation in mdx mice at 5 months of age [13, 15].
Diseases that require tissue repair, including DMD, represent a group of pathologies that have great potential for cell therapy. Currently, the use of stem cells has expanded into new areas of biotechnology and has been beneficial to the field of regenerative medicine .
Mesenchymal stem cells (MSCs) are characterised as adherent fibroblastoid cells. They have the capacity to differentiate into connective tissue cells, including adipocytes, osteocytes and myocytes, and there is evidence that MSCs can selectively differentiate in injured tissues . Among the stem cells source in adult organisms, the bulb olfactory has been studied as a potential source of multipotent stem cells in many species. For example, in rats, the fibroblast-like cells isolated from the olfactory bulb showed to express mesenchymal cells markers such as CD29 and CD90 and are also able to differentiate along osteoblastic, adipogenic and chondrogenic lineages .
Neural stem cells can be characterized on a critical functional basis in terms of their undifferentiated features, capacity for self-renewal, pluripotentiality, and ability to regenerate damaged tissue and these characteristics have been found in culture of embryonic and adult murine brain . One of the most promising sources of neural stem cells with unique characteristics are the olfactory epithelial stem cells because they are close to the higher central nervous system (CNS) and may be easily obtained in humans through a biopsy of the external nostrils .
Therefore, the aimed to assess the potential cell implantation and muscle morphology following xenotransplantation performed by the local injection of stem cells into the dystrophic diaphragm.