Abstract
Purpose: :
The extraocular muscles (EOM) have many unique properties when compared to other skeletal muscles. They are spared in muscular dystrophies, they do not age the same way as other muscles, and they remodel in the apparent absence of injury. The resilient nature of these muscles may be linked to unique properties of their satellite cells.
Methods: :
Adult rabbits or mice were sacrificed and one Tibialis anterior and 8 rectus (EOM) muscles were harvested. Muscles were enzymatically digested. Some cells were stained with Hoechst dye and sorted based on dye exclusion to be subsequently stained with antibodies against Sca–1, CD34, CD45, Pax–7, M–cadherin, Bax, and Bcl–2, others were directly stained without Hoechst sorting. Analysis was performed using flow cytometry.
Results: :
In rabbits and mice the EOM have a 5–8–fold increase in the number of satellite cells per mg muscle weight. They also are more resistant to apoptosis than cells from limb muscles of the same animals. In ex–vivo experiments, the percentage of dead cells from the limb muscle is consistently higher than from EOM. Other investigators have used Hoechst dye to separate subpopulations of precursor cells and have noted that the dye has a toxic effect. In our studies, precursor cells from the EOM are less affected by the Hoechst dye toxicity than precursor cells from the limb muscle as measured by flow cytometric analysis using 7AAD, a marker of dying or dead cells. Interestingly, other investors have used the Hoechst dye sorting method to enrich CD45+ hematopoetic stem cells, which in culture, can differentiate into a variety of tissues including muscle. Our studies show that none of the cells staining for CD45 following Hoechst sorting are viable, so it is possible that a small number of contaminating muscle satellite cells is responsible for the development of committed cells in culture. Current investigations are comparing the ratio of Bcl family member proteins within the recently extracted satellite cells of EOM and limb as a means of measuring their apoptotic potential.
Conclusions: :
It is possible that EOM remain unaffected by dystrophies and ageing because their satellite cells are resistant to apoptosis upon muscle injury, rendering them better able to regenerate and repair muscle fibers.
Keywords: extraocular muscles: development • cell survival • regeneration