Abstracts

Rationale: Oxidative stress can be a promoter or consequence of seizures. Upon brain insult, the membrane bound NADPH oxidase produces reactive oxygen species in excess. The imbalance between oxidants and antioxidants spurs neuroinflammation and hyperexcitability. Thus, the oxidative stress pathway can be a therapeutic target for epileptic seizures. Mitoapocynin (MITO) is a NADPH oxidase inhibitor that targets the mitochondria. Notably, MITO has never been tested in an animal model of epilepsy. Furthermore, the dosing regimens and pharmacokinetics (PK) of MITO in rat models have not been established. In this study, we investigated the effects of MITO (10 mg/kg) on the serum nitrooxidative stress markers and cytokines, as well as neurodegeneration and microgliosis, in a rat DFP model of epilepsy.

Methods: Adult Sprague-Dawley rats were used to test efficacy of MITO in DFP experiments. To observe the systemic distribution of MITO in the animals, healthy male rates (7-8 weeks, N=8) were given a single dose of MITO (10 mg/kg) orally. Blood samples were collected at 1 hr and 3 hr and hippocampal tissue at 3 hr. The drug concentration levels in the sera and the hippocampi were detected via LC-MS. To induce status epilepticus (SE), the randomized mixed-sex cohorts of rats (9-10 weeks, N=24) were challenged with DFP (4 mg/kg, s.c.) and immediately (~1 min) injected with 2-PAM (25 mg/kg, i.m.) and atropine sulfate (2 mg/kg, i.m.) to reduce mortality. The behavioral seizures were scored for an hour and midazolam (MDZ, 3 mg/kg, i.m.) was administered to control SE. One hour post-MDZ, the animals were treated orally with either vehicle or MITO (10 mg/kg, twice a day for 3 days and once a day for 4 days). All animals were euthanized with pentobarbital (100mg/kg, i.p.). To examine the peripheral effects of DFP and MITO, nitrite, ROS, and glutathione assays as well as key cytokines (IL-1β, IL-6, IL-10, TNF-α, and MCP1) were measured. The brain immunohistochemistry (IHC) was performed to measure neurodegeneration, microgliosis, and astrogliosis.

Results: MITO was detected in both serum (1 hr, 1939.09 pg; 3 hr, 87.06 pg) and hippocampal tissues (1 hr, 502.62 pg; 3 hr, 18.01 pg) suggesting the MITO’s serum and brain concentrations are reasonable and it crosses the blood-brain-barrier. MITO treatment mitigated the DFP-induced oxidative stress markers (nitrite, ROS, glutathione) and the proinflammatory cytokines IL-1β, IL-6, and TNF-α levels.  However, there were no differences in IL-10 and MCP1 in the serum. In DFP groups, the 10 mg/kg MITO dosing regimen was not enough to mitigate DFP-induced neurodegeneration and microgliosis.

Conclusions: At the tested dose of 10 mg/kg, MITO curtailed inflammation and oxidative stress in the periphery but not in the brain. Moreover, concentrations of the drug in the brain remain low. This implies that to achieve modifying effects, the MITO dosing regimen needs to be optimized to increase the drug concentration in the brain.

Funding: This study is funded by the NIH/NINDS CounterACT program (R21 NS120916-01).