Evaluation of the neuroprotective activity of gamma‐benzylidenes digoxin derivatives against brain ischemia

Abstract

Brain ischemia is one of the diseases with the highest mortality rate today. Among the victims who do not die, most will have some functional disability as a sequel. Currently, tissue plasminogen activator (rt‐PA) is the only pharmacological treatment for ischemia and there are no treatments with neuroprotective effects for this disease. Recently, cardiotonic steroids (CTS) have been shown to promote neuroprotection against ischemia. However, CTS are drugs that have a narrow therapeutic index and their utilization in clinic are restrict. BD‐15 is a new a non‐toxic benzylidine digoxin derivative that was demonstrated to direct increase of the α3‐Na,K‐ATPase activity, and to prevent chemical ischemia in N2a cells model, but it was never tested in a ischemia model in animal. We report here the results of the use of BD‐15 against cognitive damage and neurotoxic actions caused during brain ischemia. Wistar rats were divided in 4 groups (6 animals per group): Control, Control + BD 100 µg/kg treatment, Ischemia and Ischemia + BD‐15 100 µg/kg treatment. Transient brain ischemia was induced with bilateral occlusion of both common carotid arteries for 30 minutes, and after that the animal were followed by three days of reperfusion, with or without BD‐15 treatment (one dose per day IP, 100 µg/kg) and the hippocampus was collected to perform biochemical analysis such as: membrane lipid profile and peroxidation and membrane enzyme activities (PMCA, SERCA, and acetylcholinesterase); histological analysis for infarct volume quantification using TTC dye, and behavioral parameters assessed by the open field behavior test (the parameters evaluated were the number of times the animal changed quadrant, raised its body and presented self‐cleaning behavior and the number of visits to the central quadrants) were also evaluated. In histological analysis, the ischemia provoked a brain tissue damage after 3 days reperfusion, and BD‐15 treatment was able to impair this damage. Ischemia provoked an decrease in the total Na,K‐ATPase activity and an increase of the expression levels of the pump and BD‐15 prevent that changes caused by ischemia. The same profile was found for total PMCA activity and PMCA4 expression levels. BD‐15 was also able to prevent the decrease of activity of SERCA, Mg‐ATPase and acetylcholinesterase caused by ischemia. The membrane lipid peroxidation (thiobarbituric acid reactive substance), was increased in the ischemic group, and again BD‐15 was able to prevent this effect. Regarding the lipid profile, we observed an increase in the content of phospholipids and cholesterol in the ischemic group, and an decrease in the groups treated with BD‐15. The behavioral parameters demonstrated that BD‐15 prevents the motor damage caused by ischemia. In conclusion, BD‐15 is a non‐toxic CTS that demonstrate the prevention of brain damages provoked by ischemia, and appear to promote neuroprotection in animals exposed to global ischemia.