Dose-response, therapeutic time-window and tPA-combinatorial efficacy of compound 21: A randomized, blinded preclinical trial in a rat model of thromboembolic stroke

Abstract

The aim of this translational, randomized, controlled, blinded preclinical trial was to determine the effect of compound 21 (C21) in embolic stroke. Rats were subjected to embolic-middle cerebral artery occlusion (eMCAO). They received C21 (0.01, 0.03 and 0.06 mg/kg/d) or saline (orally) for five days, with the first-dose given IV at 3 h post-eMCAO. For the time-window study, the optimal-dose of C21 was initiated at 3, 6 or 24 h post-eMCAO and continued for five days. For the combinatorial study, animals received IV-tissue plasminogen activator (tPA) at either 2 or 4 h, with IV-C21 (0.01 mg/kg) or saline at 3 h post-eMCAO and daily thereafter for five days. After performing the behavior tests, brains were collected for analyses. The dose-response study showed significant motor improvements with the lowest-dose (0.01 mg/kg) of C21. In the time-window study, this same dose resulted in improvements when given 6 h and 24 h post-eMCAO. Moreover, C21-treated animals performed better on the novel object recognition test. Neither the single treatment with C21 or tPA (4 h) nor the combination therapy was effective in reducing the hemorrhage or infarct size, although C21 alone lowered sensorimotor deficit scores post-eMCAO. Future studies should focus on the long-term cognitive benefits of C21, rather than acute neuroprotection.

Keywords: Compound C21; embolic middle cerebral artery occlusion; functional outcome; ischemic stroke; tissue plasminogen activator.

Figure 1.

Schematic description of experimental design with animal group numbers.

Figure 2.

Dose–response effect of C21 on functional outcome after eMCAO (a–g). Motor function tests were performed at days 1, 3, 5 and 7 after stroke. Repeated-measures ANOVA showed significant improvement with the low dose (0.01 mg/kg) of C21 on functional outcomes in Bederson (*p < 0.05 vs. 0.03 dose, ^#p < 0.05 vs. saline and 0.03 dose) (a), Bederson incorporating mortality (b), paw grasp (c), beam walk (*p < 0.05 vs. 0.03 dose, ^#p < 0.05 saline vs. 0.03 dose) (d), and grip strength (*p < 0.05 vs. saline) (e) tests after stroke. This improvement was seen at days 3, 5 or 7, and shows a neurorestorative effect. Statisticians Guidance on “Superior” Group – From the plots and the statistical analysis it appears that low dose (0.01 mg/kg) of C21 is superior. There were no statistically significant differences between saline-treated and C21-treated groups for weight (f) and percent infarct size (g). Values are expressed as mean ±SD and median ± 25th and 75th percentiles.

Figure 3.

Dose–response effect of C21 on NOR at seven days after eMCAO. NOR was performed at seven days, prior to sacrifice. The discrimination index is the time spent with the novel object – time spent with the familiar object/ total time (done by video collection). A positive number indicates more time spent with the novel object. As can be seen, the saline-treated animals showed no preference for the novel object, whereas the animals treated with C21, seemed to remember the familiar object and spend more time with the novel object. NOR: novel object recognition. Values are expressed as mean ±SD.

Figure 4.

Therapeutic time window of C21 on functional outcome after eMCAO (a–f). Motor function tests were performed at days 1, 3, 5 and 7 after stroke. C21 (0.01 mg/kg) group exhibited improvement in Bederson score (a) with 3, 6 and 24 h delays after stroke onset (*p < 0.05 saline vs. 6 h, ^#p < 0.05 saline vs. 24 h). Compared with placebo, compound C21 showed a significant improvement in Bederson score (a) as late as 6 h and 24 h after stroke. There were no statistically significant differences between groups in Bederson with mortality (b), paw GRASP (c), beam walk (d), grip strength (e) and weight (f) between saline-treated and C21-treated groups. Statisticians Guidance on “Superior” Group – There is no statistical significance. From the plots, it appears that 6 h and 24 h groups are superior to saline. Values are expressed as mean ± SD and median ± 25th and 75th percentiles.

Figure 5.

C21 in combination with tPA (2 h) for 28 days survival after eMCAO (a–j). Motor function tests were performed at days 7, 14, 21 and 28 after stroke. Combination therapy with tPA did not show any benefit in hemorrhage, weight (a), Bederson (b), Bederson incorporating mortality (c), paw grasp (d), beam walk (e) and grip strength (f) over time. In the rotarod (g, h), tPA group only showed decrease in time from day 0 to day 1 and then little to no improvement from days 7 to 28. There were no differences between groups within rotarod measurement days. There was not a statistically significant interaction between all groups and day for NOR DI at 3 min (i) or 5 min (j). C21 group only exhibited significant improvement in NOR DI at 5 min (j) than group C21 + tPA on day 28 after eMCAO (*p < 0.05). Values are expressed as mean ±SD and median ± 25th and 75th percentiles.