The Roles of cell death inhibitors on brain pathologies after cardiac Ischemia/Reperfusion injury in rats

Abstract

Recently, the association between cardiac dysfunction and central nervous system (CNS) injury is of great investigational interest. Despite effective reperfusion therapy, patients with myocardial infarction have been reported to have cognitive impairment. Several types of cell death are involved in brain injury during cardiac ischemia reperfusion (IR) injury. Although inhibitors of apoptosis, necroptosis, and ferroptosis provided neuroprotection against cerebral I/R injury, their effects on the brain following cardiac I R injury have never been investigated. Three hypotheses were tested in this thesis: 1) cardiac I/R injury led to brain cell death including apoptosis. necroptosis. and ferroptosis. 2) cell death inhibitors reduced brain pathology by reducing mitochondrial dysfunction. inflammation, and Alzheimer's disease-related protein. thereby preserving dendritic spine density following cardiac I R injury, and 3) combined treatment provided better neuroprotective effects than single treatment regimen. To test the first and second hypotheses, one-hundred and twenty-six male rats were used: 6 rats were assigned to the sham operation and 120 rats were subjected to 30-min regional cardiac ischemia by ligation of the left anterior descending coronary artery, followed by 120-min reperfusion. Rats in the cardiac I/R group were pretreated with either a vehicle (normal saline solution, n=12) or one of the cell death inhibitors (apoptosis, necroptosis, or ferroptosis inhibitor). Z-VAD. necrostatin-1 (Nec-1), and ferrostatin-1 (Fer-1) were used as inhibitors of apoptosis, necroptosis, and ferroptosis, respectively. Rats in each treatment group were subdivided into three different doses including low (1.65 mg/kg for Z-VAD and Nec-1, 1 mg/kg for Fer-1), medium (3.3 mg/kg for Z-VAD and Nec-1, 2 mg/kg for Fer-1), and high doses (6.6 mg/kg for Z-VAD and Nec-1, 4 mg/kg for Fer-1). The number of rats in each group was 12. Rats were sacrificed at the end of reperfusion, and the brains of rats were used to analyze mitochondrial function, blood-brain barrier (BBB) tight junction proteins, dendritic spine density, Alzheimer's disease (AD)-related proteins, neuroinflammation. and cell death. Our data showed that cardiac I/R injury caused brain damage including brain inflammation and mitochondrial dysfunction, and we found that only apoptosis occurred in the hippocampus after cardiac I/R injury. In the cardiac I/R group, treatment with Z-VAD at the medium dose and all doses of Nec-1 decreased hippocampal apoptosis, amyloid-beta aggregation, and they were associated with a decrease in dendritic spine loss. Treatment with Fer-1 at medium and high doses also reduced dendritic spine loss by suppressing ACSL4, TNF-α. amyloid-beta, and tau hyperphosphorylation. Moreover, Bax/Bcl-2 ratio was decreased in all treatment regimen except Z-VAD treatment at the low dose. Additionally, treatment with Z-VAD and Fer-1 at the medium dose partially attenuated mitochondrial dysfunction. Only treatment with the low dose of Nec-1 preserved BBB tight junction protein. Therefore, our data indicated that cell death inhibitors prevented hippocampal dendritic spine loss caused by cardiac I/R injury through different mechanisms. In addition to the neuroprotective effects, medium and high dose of Z-VAD and Nec-1 reduced myocardial infarction. Regarding these data, medium dose of Z-VAD and Fer-1 were selected to be used as a combination treatment to test whether the combined therapy provided better neuroprotective effects in rats with cardiac I/R injury than a single treatment regimen. Thirty rats with cardiac I/R injury were pretreated with combined Z-VAD at 3.3 mg/kg and Fer-1 at 2 mg/kg. A comparative analysis of combined treatment and single treatment was performed. Rats were sacrificed at the end of reperfusion, and the brains of rats were used to measure dendritic spine density. Alzheimer's disease (AD)-related proteins. blood-brain barrier BBB) tight junction proteins. mitochondrial function. inflammation. and cell death. Unexpectedly, our results showed that combined treatment exerted no synergistic effects on dendritic spine density, AD protein expression such as Aβ /APP protein levels. BACE1 protein level, and tau hyperphosphorylation. and inflammation. We also found that combined treatment effectively reduced apoptosis, but it did not affect ferroptosis and necroptosis in rats with cardiac I/R injury, suggesting that apoptosis plays a crucial role in regulating brain injury in this setting. Regarding mitochondrial function. although ROS did not further decrease by the combined treatment. mitochondrial swelling was greatly reduced in the combined group than in the single treatment. In summary, our data indicated that cell death inhibitors attenuated hippocampal dendritic spine loss caused by cardiac I/R injury through different mechanisms. Z-VAD promoted neuroprotection by ameliorating apoptosis and mitochondrial dysfunction. Nec-1 preserved BBB proteins and reduced apoptosis, an effect that is beyond its RIPK1 inhibition, while Fer-1 suppressed ACSL4. All treatment effectively reduced AD-related proteins and tau hyperphosphorylation in the brain following cardiac I/R injury. Although medium-dose Z-VAD and Fer-1 provided the best efficacy in reducing brain damage, the synergistic effects of medium-dose of Z-VAD and Fer-1 were not observed in the combined treatment group.