Resumo

Purpose: Amyotrophic lateral sclerosis (ALS) is a adult-onset neurodegenerative disorder characterized by degeneration and loss of motor neurons in the cerebral cortex, brainstem, and spinal cord leading to muscle wasting and weakness and eventually to death within 5 years after the onset of clinical symptoms. There is no effective treatment because the mechanisms involved in the disease are unknown. Recent publications have put non-neuronal cells, particularly, astrocyte and microglia, in the scenario of pathophysiology of the disease. Animal models for ALS, particularly transgenic mice G93A are available and display the phenotype of the disease at cellular and clinical levels. However, is a lack of detailed information regarding the methods to study the disease in vitro that provide a better understand the contribution of non-neuronal cells in the onset and progression of the pathology. The available publications does not specify the methods carefully. Methods: The G93A mice identification as transgenic or wild-type (control) in our colony was performed by a genotyping method. The literature was revised in order to find descriptions of highly purified cultures of glial cell types and neurons. We attempted to detail methods to achieve highly purified astrocyte, microglia and motor neurons from spinal cord of ALS mice. Cells were identified by means of glial fibrilary acid protein, CD11B and choline acetyltransferase immunocytochemistry. The purity of cell cultures was also accompanied by means of western blot and reverse transcriptase polimerase chain reaction (RT-PCR) analyses employing a number of markers to make sure that the cultures were purified. Results: Immunocytochemical, biochemical and RT-PCR analyses demonstrated highly purified primary cultures of spinal cord astrocytes, microglia and motor neurons leading a study of the individual behavior cell in ALS. The astrocyte and microglia culture were treated with trypsin, shaking and cytosine arabinoside (Arac) as a purification strategy for elimination other glial cells that could be contaminating the cultures. The same happened for the motor neuron culture which was separated by density ensuring the purity. Then we are sure that our cultures are purified and do not contains influences from other cells, thus becoming a good object of study for ALS. Conclusion: These results suggest that it is possible to achieve highly purified primary cultures of spinal cord astrocytes, microglia and motor neurons to be employed in cellular and molecular analyses of the influence of those cells in the pathophysiology of ALS. All together, these advantages imply that co culture of purified glial and neuron cell could be used as a solid platform for studies of the disease.