Tempol Moderately Extends Survival in a hSOD1G93A ALS Rat Model by Inhibiting Neuronal Cell Loss, Oxidative Damage and Levels of Non-Native hSOD1G93A Forms
Linares E¹, Seixas LV, dos Prazeres JN, Ladd FV, Ladd AA, Coppi AA, Augusto O.
¹Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive dysfunction and death of motor neurons by mechanisms that remain unclear. Evidence indicates that oxidative mechanisms contribute to ALS pathology, but classical antioxidants have not performed well in clinical trials. Cyclic nitroxides are an alternative worth exploring because they are multifunctional antioxidants that display low toxicity in vivo. Here, we examine the effects of the cyclic nitroxide tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) on ALS onset and progression in transgenic female rats over-expressing the mutant hSOD1(G93A) . Starting at 7 weeks of age, a high dose of tempol (155 mg/day/rat) in the rat´s drinking water had marginal effects on the disease onset but decelerated disease progression and extended survival by 9 days. In addition, tempol protected spinal cord tissues as monitored by the number of neuronal cells, and the reducing capability and levels of carbonylated proteins and non-native hSOD1 forms in spinal cord homogenates. Intraperitoneal tempol (26 mg/rat, 3 times/week) extended survival by 17 days. This group of rats, however, diverted to a decelerated disease progression. Therefore, it was inconclusive whether the higher protective effect of the lower i.p. dose was due to higher tempol bioavailability, decelerated disease development or both. Collectively, the results show that tempol moderately extends the survival of ALS rats while protecting their cellular and molecular structures against damage. Thus, the results provide proof that cyclic nitroxides are alternatives worth to be further tested in animal models of ALS.
Effects of oral tempol on neuron degeneration in the lumbar spinal cord from G93A rats. Representative light-microscopic images of toluidine-blue stained (A, A-I, B, B-I, C and C-I) and ChAT immunolabeled (A-II, B-II and C-II) sections of the lumbar spinal cord from wild-type and G93A rats both untreated and treated with oral tempol. Female rats received tempol (30 mM) in their drinking water starting at 7 weeks of age and were sacrificed at the time of middle symptomatic phase of G93A rats (115–120 days). A) wild-type rats; B) untreated G93A rats; and C) treated G93A rats. White matter (1), grey matter (2), ventral horn (3) and the central canal (*) are labeled as specified. Also, note the toluidine blue-stained (arrows in A-I, B-I and C-I) and immunolabeled ChAT-positive (arrowheads in A-II, B-II and C-II) neurons. Scale bars: 100 µm (A, B and C) and 20 µm (A-I, B-I, C-I, A-II, B-II and C-II). Stereological methods were conducted in 15 rats, 5 animals per each group: wild-type, untreated G93A and treated G93A. Ten 40 µm-thick systematic, uniform and random sections were analyzed per animal, summing up 50 sections per group and 150 sections in the 3 studied groups.