Simvastatin improves clinical scores in a rabbit multiple infarct ischemic stroke model: synergism with a ROCK inhibitor but not the thrombolytic tissue plasminogen activator

Abstract

Statins have pleiotropic neuroprotective effects in the central nervous system. In this study, we assessed the pharmacological effects of simvastatin on measures of behavior in New Zealand white rabbits embolized using a suspension of small-sized blood clots. For these studies, simvastatin was administered up to 3 hours following embolization, and behavior was measured 48 hours following embolization to calculate the dose of emboli (P(50) in mg) that produces neurological deficits in 50% of the rabbits. A treatment is considered neuroprotective if it significantly increases the P(50) compared to control. Simvastatin treatment (20mg/kg, bolus subcutaneous injection) significantly improved clinical function and increased the P(50) by 143% when administered 1 hour following embolization but was ineffective at 3 hours. In combination studies with the thrombolytic, tissue plasminogen activator (tPA) using a standard intravenous dose of 3.3mg/kg (20% bolus, 80% infused), we found that simvastatin could be safely administered with tPA to improve clinical scores; however, the maximum behavioral improvement with the combination treatment was similar to either monotherapy alone, both of which significantly improved behavior (p<0.05). It has been proposed that Simvastatin neuroprotection may be related to a variety of signaling pathways including Rho-kinase (ROCK). To determine if a ROCK mechanism is involved in simvastatin-induced neuroprotection following embolic strokes, we used pharmacological intervention with the ROCK inhibitor, fasudil. When fasudil was administered 30 minutes before simvastatin (given at 1 hour), there was an additional significant (p=0.0217) synergistic increase in behavioral function. However, fasudil as a monotherapy did not affect behavioral function in embolized rabbits. The study suggests that there may be an interaction between simvastatin treatment and the ROCK signaling pathway that should be further explored. Our results suggest that simvastatin treatment may have clinical benefit when used alone or in the presence of tPA, but the therapeutic window using a single-dose regimen is narrow.

Figure 1. Quantal Curves

Effect of simvastatin (20 mg/kg) on clinical function or behavior measured following embolization. For the superimposed graphs, normal animals are plotted on the y-axis at 0 and abnormal animals are plotted at 100. The figure shows that there is positive correlation between the data [circles (vehicle) or triangles (simvastatin)] and the statistically fit sigmoidal quantal curve. Statistical significance (*p<0.05).

Figure 2. Therapeutic Window

P₅₀ (mg clot in brain) as a function of Time post-embolization (minutes). Results are shown as mean ± SEM (n= 20–32). Significantly different compared to the vehicle-treated control (*p<0.05).

Figure 3. Combination Analysis- Simvastatin & tPA

Abnormal rabbits as a function of clot weight measured in brain. Results are shown as mean ± SEM for the number of rabbits in each group (n). Both simvastatin (20 mg/kg) and tPA (3.3mg/kg) treatment 1 hour post-embolization significantly (*p<0.05) increased P₅₀ values. The combination of simvastatin (1 hour) plus tPA (1 hour) significantly (*p<0.05) improved behavior and increased P₅₀ values compared to control. The combination was not significantly different (p<0.05) from either simvastatin or tPA monotherapy.

Figure 4. Combination Analysis- Mechanism of Action

Abnormal rabbits as a function of clot weight measured in brain. Results are shown as mean ± SEM for the number of rabbits in each group (n). Simvastatin (20 mg/kg) treatment 1 hour post-embolization significantly (*p<0.05) increased P₅₀ values compared to control. The ROCK inhibitor FASUDIL (HA-1077, 10 mg/kg) was ineffective (p>0.05) as a monotherapy. The combination of simvastatin plus FASUDIL, both administered 1 hour following embolization significantly improved behavior and increased P₅₀ values compared to control(*p<0.05) and there was a synergistic effects of the combination therapy (#p=0.0217 compared to simvastatin).