Beta-lactam antibiotic offers neuroprotection in a spinal muscular atrophy model by multiple mechanisms

Histological analysis of muscles of hind limbs for total tibialis anterior (TA), cross sectional area, total TA myofiber number and myofiber diameter was performed by the author.Spinal cord sections were subjected to immunohistochemistry using monoclonal antibodies against motoneuron specific markers SMI 32 antigen followed by secondary antibody AlexFlour 488.Purified RNA isolated from spinal cord followed by cRNA preparation and oligonucleotide microarray hybridization.Biotinylated cRNA was hybridized to Affymetrix GeneChip Mouse Genome 430A 2.0 array at 47 o C overnight and visualized on GeneArray 2500 Scanner.RNA from four groups of selected animals was reverse transcribed on Real time PCR system.Spinal cord protein was extracted from experimental animals and subjected to semi quantitative western blot.The membranes were probed with mouse anti SMN, anti EAAT2 antibody, rabbit anti BAX, anti PRG2, anti TDP 43, anti FUS and anti actin antibodies followed by use of secondary antibodies and chemiluminescence detection techniques.The staining sections and proteins density were measured with NIH Image software.Kaplan Meier analysis was used on lifespan using log rank post hoc test.ANOVA and Tukey-post hoc analysis of growth curve was performed.Student's test was used for statistical analysis and upper significance level of 0.05 was used.