Adult aberrant astrocytes submitted to late passage cultivation lost differentiation markers and decreased their pro-inflammatory profile.

In amyotrophic lateral sclerosis (ALS), astrocytes are considered key players in some non-cell non-neuronal autonomous mechanisms that underlie motor neuron death. However, it is unknown how much of these deleterious features were permanently acquired. To assess this point, we evaluated if the most remarkable features of neurotoxic aberrant glial phenotypes (AbAs) isolated from paralytic rats of the ALS model G93A Cu/Zn superoxide dismutase 1 (SOD1) could remain upon long lasting cultivation. Real time PCR, immunolabelling and zymography analysis showed that upon many passages, AbAs preserved the cell proliferation capacity, mitochondrial function and response to different compounds that inhibit some key astrocyte functions but decreased the expression of parameters associated to cell lineage, homeostasis and inflammation. As these results are contrary to the sustained inflammatory status observed along disease progression in SOD1G93A rats, we propose that the most AbAs remarkable features related to homeostasis and neurotoxicity were not permanently acquired and might depend on the signaling coming from the injuring microenvironment present in the degenerating spinal cord of terminal rats.

Neuroprotective effects of violacein in a model of inherited amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive death of motor neurons and muscle atrophy, with defective neuron-glia interplay and emergence of aberrant glial phenotypes having a role in disease pathology. Here, we have studied if the pigment violacein with several reported protective/antiproliferative properties may control highly neurotoxic astrocytes (AbAs) obtained from spinal cord cultures of symptomatic hSOD1G93A rats, and if it could be neuroprotective in this ALS experimental model. At concentrations lower than those reported as protective, violacein selectively killed aberrant astrocytes. Treatment of hSOD1G93A rats with doses equivalent to the concentrations that killed AbAs caused a marginally significant delay in survival, partially preserved the body weight and soleus muscle mass and improved the integrity of the neuromuscular junction. Reduced motor neuron death and glial reactivity was also found and likely related to decreased inflammation and matrix metalloproteinase-2 and -9. Thus, in spite that new experimental designs aimed at extending the lifespan of hSOD1G93A rats are needed, improvements observed upon violacein treatment suggest a significant therapeutic potential that deserves further studies.

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions.

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.