Cell Biology

Cell transplantation is a promising strategy for treating age-related muscle atrophy, but its critical application remains limited. Cultured myoblasts, unlike freshly isolated muscle stem cells, show poor engraftment efficiency and fail to contribute effectively to muscle regeneration. Moreover, successful engraftment generally requires prior muscle injury, as skeletal muscle regeneration is typically triggered by a damaged microenvironment. These limitations present major obstacles for applying cell therapy to sarcopenia, where muscle degeneration occurs without injury. In this protocol, we describe a novel approach that enables the transplantation of cultured myoblasts into intact skeletal muscle without the need for preexisting injuries or genetic modification. By combining myoblasts with extracellular matrices (ECM), such as Matrigel, which mimic the native muscle niche and support cell survival, adhesion, proliferation, and differentiation, we achieve efficient engraftment and increased muscle mass without the need for preexisting injury. The ECM also provides a scaffold and retains bioactive factors that enhance the regenerative capacity of transplanted cells. This is the first protocol that enables robust myoblast engraftment in non-injury muscle conditions, offering a practical tool for studying and potentially treating sarcopenia.

The neuromuscular junction (NMJ) is critical for muscle function, and its dysfunction underlies conditions such as sarcopenia and motor neuron diseases. Current protocols for assessing NMJ function often lack standardized stimulation parameters, limiting reproducibility. This study presents an optimized ex vivo method to evaluate skeletal muscle and NMJ function using the Aurora Scientific system, incorporating validated stimulation protocols for both nerve and muscle to ensure consistency. Key steps include tissue preparation in a low-calcium, high-magnesium solution to preserve NMJ integrity, determination of optimal muscle length, and sequential stimulation protocols to quantify neurotransmission failure and intratetanic fatigue. By integrating rigorous standardization, this approach enhances reproducibility and precision, providing a robust framework for investigating NMJ pathophysiology in aging and disease models.

The lumen of blood vessels is covered by endothelial cells, which regulate their permeability to ions and solutes. Endothelial permeability depends on the vascular bed and cell phenotype, and is influenced by different disease states. Most characterization of endothelial permeability has been carried out using isolated cells in culture. While analysis of cultured cells is a valuable approach, it does not account for factors of the native cell environment. Building on Ussing chamber studies of intact tissue specimens, here we describe a method to measure the electrophysiological properties of intact arteriole and venule endothelia, including transendothelial electrical resistance (TEER) and ion permselectivity. As an example, vessels isolated from the mesentery were treated ex vivo, then mounted in a custom-made tissue cassette that enable their analysis by classical approaches with an Ussing chamber. This method enables a detailed analysis of electrophysiological vessel responses to stresses such as proinflammatory cytokines, in the context of an intact vessel.

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According to the World Health Organization (WHO), nearly 1.13 billion people worldwide have hypertension, a major factor responsible for premature death globally. The inherent multifactorial nature of hypertension makes its study difficult since the chronic rise in blood pressure depends on the intricate connection between dietary, genetic and environmental factors. Therefore, the pathophysi-ology of hypertension is not completely understood. For these reasons, there is an ongoing search for animal models that better mimic changes resulting from this disease. Because of its complexity, the use of animal models aimed at elucidating the pathogenesis of hypertension and to evaluate new therapeutic possibilities is an important tool for understanding this disease since it enables consistent experimental strategies that are impractical in humans. Over time, many animal models have been developed for the study of chronic increases in blood pressure ranging from genetic models that include the spontaneously hypertensive rat (SHR) and genetic manipulations, such as the TGR (mRen2) rat, as well as neurogenic or endocrine models. One of the most commonly used hypertensive rat models today is that of hypertension induced by treatment with deoxycorticosterone acetate associated with high sodium intake, i.e., the DOCA-salt model. This model is known to have a neurogenic component linked to increased sympathetic nervous system activity, and as such the DOCA-salt model promotes cross-talk between endocrine and neural components that lead to increased blood pressure, and may impact the functioning of other organs.

The neuromuscular junction (NMJ) is the specialized synapse by which peripheral motor neurons innervate muscle fibers and control skeletal muscle contraction. The NMJ is the target of several xenobiotics, including chemicals, plant, animal and bacterial toxins, as well as of autoantibodies raised against NMJ antigens. Depending on their biochemical nature, the site they target (either the nerve or the muscle) and their mechanism of action, substances affecting NMJ produce very specific alterations of neuromuscular functionality.

Here we provide a detailed protocol to isolate the diaphragmatic muscle from mice and to set up two autonomously innervated hemidiaphragms. This preparation can be used to study bioactive substances like toxins, venoms and neuroactive molecules of various origin, or to measure the force of skeletal muscle contraction.

The ‘mouse phrenic nerve hemidiaphragm assay’ (MPN) is an established model of ex vivo NMJ and recapitulates the complexity of neuromuscular transmission in a system easy to control and to manipulate, thus representing a valuable tool to study both NMJ physiology and the mechanism of action of toxins and other molecules acting at this synapse.

Although it is known that the generation of movements is performed to a large extent in neuronal circuits located in the spinal cord, the involved mechanisms are still unclear. The turtle as a model system for investigating spinal motor activity has advantages, which far exceeds those of model systems using other animals. The high resistance to anoxia allows for investigation of the fully developed and adult spinal circuitry, as opposed to mammals, which are sensitive to anoxia and where using neonates are often required to remedy the problems. The turtle is mechanically stable and natural sensory inputs can induce multiple complex motor behaviors, without the need for application of neurochemicals. Here, we provide a detailed protocol of how to make the adult turtle preparation, also known as the integrated preparation for electrophysiological investigation. Here, the hind-limb scratch reflex can be induced by mechanical sensory activation, while recording single cells, and the network activity, via intracellular-, extracellular- and electroneurogram recordings. The preparation was developed for the studies by Petersen et al. (2014) and Petersen and Berg (2016), and other ongoing studies.