A New NF-κB Inhibitor, MEDS-23, Reduces the Severity of Adverse Post-Ischemic Stroke Outcomes in Rats

Abstract

Aim: Nuclear factor kappa B (NF-κB) is known to play an important role in the inflammatory process which takes place after ischemic stroke. The major objective of the present study was to examine the effects of MEDS-23, a potent inhibitor of NF-κB, on clinical outcomes and brain inflammatory markers in post-ischemic stroke rats.

Main methods: Initially, a Toxicity Experiment was performed to determine the appropriate dose of MEDS-23 for use in animals, as MEDS-23 was analyzed in vivo for the first time. We used the middle cerebral artery occlusion (MCAO) model for inducing ischemic stroke in rats. The effects of MEDS-23 (at 10 mg/kg, ip) on post-stroke outcomes (brain inflammation, fever, neurological deficits, mortality, and depression- and anxiety-like behaviours) was tested in several efficacy experiments.

Key findings: MEDS-23 was found to be safe and significantly reduced the severity of some adverse post-stroke outcomes such as fever and neurological deficits. Moreover, MEDS-23 significantly decreased prostaglandin E2 levels in the hypothalamus and hippocampus of post-stroke rats, but did not prominently alter the levels of interleukin-6 and tumor necrosis factor-α.

Significance: These results suggest that NF-κB inhibition is a potential therapeutic strategy for the treatment of ischemic stroke.

Keywords: NF-κB; depression; inflammation; ischemic stroke; mortality; stroke.

Figure 1

Effects of MEDS-23 treatment on BW of post-stroke rats. Sham- and MCAO-operated rats were treated with vehicle (DMSO 0.1 mL/rat) or MEDS-23 10 mg/kg for a total of 15 days through a single daily injection (i.p.). BW was measured every day before treatment. The figure represents the combined results of all four Efficacy Experiments. Data are presented as percentage of baseline BW, which is expressed as 100%. Each point represents mean ± SEM of a different number of rats in each group at the various time-points of the experiment, as shown in Table S5. Using dependent samples t-test—* p < 0.05 vs. Sham + Vehicle—at the same time point. MCAO—middle cerebral artery occlusion; Veh—vehicle.

Figure 2

Effects of MEDS-23 treatment on BT of post-stroke rats. Sham- and MCAO-operated rats were treated with vehicle (DMSO 0.1 mL/rat) or MEDS-23 10 mg/kg for a total of 15 days through a single daily injection (i.p.). BT was measured before and at 24, 48 and 72 h post-surgery. The figure represents the results of a single efficacy experiment (very similar results were obtained in other experiments). Each point represents mean ± SEM of a different number of rats in each group at the various time-points of the experiment. At baseline, the sample size in each group was as follows: Sham + Vehicle = 19, Sham + MEDS-23 = 19, MCAO + Vehicle = 30, MCAO + MEDS-23 = 31. Using two-way ANOVA—* p < 0.05 vs. Sham + Vehicle, ^ p < 0.05 vs. Sham + MEDS-23, # p < 0.05 vs. MCAO + Vehicle—at the same time-point. MCAO—middle cerebral artery occlusion; Veh—vehicle.

Figure 3

Effects of MEDS-23 treatment on brain inflammatory mediator levels in post-stroke rats. Sham- and MCAO-operated rats were treated with vehicle (DMSO 0.1 mL/rat) or MEDS-23 10 mg/kg for a total of 15 days through a single daily injection (i.p.). Brain regions were extracted as described in “Materials and Methods” at 14 days after surgery. PGE2 (A–C), IL-6 (D–F) and TNF-α (G–I) levels in HT (A,D,G), HC (B,E,H) and FC (C,F,I) were determined by specific ELISA kits. Each column is the mean ± SEM of 10 to 21 samples per group. Using two-way ANOVA test (two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli)—* p < 0.05 vs. Sham + Veh; ^ p < 0.05 vs. Sham + MEDS-23; # p < 0.05 vs. MCAO + Veh—between-group comparisons. FC—frontal cortex, HC—hippocampus, HT—hypothalamus, IL-6—interleukin-6, MCAO—middle cerebral artery occlusion, PGE2—prostaglandin E2, TNF—tumor necrosis factor, Veh—vehicle.

Figure 4

Effects of MEDS-23 treatment on NS of post-stroke rats. Sham- and MCAO-operated rats were treated with vehicle (DMSO 0.1 mL/rat) or MEDS-23 10 mg/kg for a total of 15 days through a single daily injection (i.p.). NS was assessed as described in “Materials and Methods” before and at 2, 24, 48 and 72 h post-surgery. The figure represents the combined results of all four Efficacy Experiments. Each point represents mean ± SEM of a different number of rats in each group at the various time-points of the experiment as shown in Table S5. Using multiple t-test—* p < 0.05 vs. Sham + Vehicle, ^ p < 0.05 vs. Sham + MEDS-23, # p < 0.05 vs. MCAO + Vehicle—at the same time-point. MCAO—middle cerebral artery occlusion; Veh—vehicle.

Figure 5

Effects of MEDS-23 treatment on mortality of post-stroke rats. Sham- and MCAO-operated rats were treated with vehicle (DMSO 0.1 mL/rat) or MEDS-23 10 mg/kg for a total of 15 days through a single daily injection (i.p.). Rat mortality was monitored for 14 days post-surgery. The figure represents the combined results of all four Efficacy Experiments. Survival rates at the various time-points of the experiment are presented as percentage of baseline survival which is expressed as 100%. The sample size in each group at baseline was as follows: Sham + Vehicle = 30, Sham + MEDS-23 = 31, MCAO + Vehicle = 53, MCAO + MEDS-23 = 54. An independent samples log-rank (Mantel-Cox) test revealed significant survival estimates between the groups (p = 0.028). Fisher-exact test was used to compare the difference between particular groups—* p< 0.05 vs. Sham + Vehicle, ^ p < 0.05 vs. Sham + MEDS-23—at the same time-point. MCAO—middle cerebral artery occlusion, Veh—vehicle.