Protective effects of statins involving both eNOS and tPA in focal cerebral ischemia

Abstract

Previous studies have shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) protect the brain against ischemic injury by upregulating endothelial nitric oxide synthase (eNOS). Here, we tested the hypothesis that statins provide additional beneficial effects by also upregulating endogenous tissue plasminogen activator (tPA) and enhancing clot lysis in a mouse model of embolic focal ischemia. Heterologous blood clots (0.2 mm) were injected into the distal internal carotid artery to occlude blood flow in the middle cerebral artery territory after long-term (14 days) simvastatin, atorvastatin or vehicle treatment. Ischemic lesion volume, neurologic deficits, as well as residual blood clots were measured at 22 h. Reverse transcription-polymerase chain reaction assessed mRNA levels of eNOS, tPA, and the endogenous plasminogen activator inhibitor PAI-1. Ischemic lesion volumes and neurologic deficits were significantly reduced in wild-type mice by both simvastatin and atorvastatin. Statins increased eNOS and tPA mRNA levels but did not change mRNA levels of PAI-1. In eNOS knockout mice, atorvastatin reduced the volume of ischemic tissue and improved neurologic outcomes after arterial occlusion by blood clot emboli. In contrast, statins did not have protective effects in tPA knockout mice after embolic focal ischemia, but only in a filament model where focal ischemia was achieved via mechanical occlusion. These results suggest that statins protect against stroke by multiple mechanisms involving both eNOS and tPA. The involvement of each pathway may be revealed depending on the choice of experimental stroke model.

Figures

Figure 1

(A) 24 hr ischemic lesion volumes (mean+SEM) in SV-129 mice after embolic focal ischemia were significantly reduced (*P

Figure 2

(A) The effects of statins on the clot itself was assessed by using clots derived from atorvastatin treated donors (n=10) or vehicle treated donors (n=10) and comparing ischemic lesion volumes (mean+SEM) in untreated SV-129 mice. No protective effect was observed when clots derived from atorvastatin treated donors were used in untreated mice. U/V = untreated mice occluded with clots from vehicle treated donors. U/A = untreated mice occluded with clots from atorvastatin treated donors. (B) 24 hr ischemic lesion volumes (mean+SEM) in SV-129 mice after embolic focal ischemia were significantly reduced (*P

Figure 3

Representative photographs of the residual clots in the middle cerebral arteries of ischemic brains at 24 hrs. Atorvastatin treated mice had significantly reduced residual clot compared with vehicle treated controls (see Table 3 for quantified data).

Figure 4

(A) Representative semi-quantitative RT-PCR for eNOS. (B) Densitometric analysis of eNOS mRNA levels normalized to beta-actin levels (mean+SEM). eNOS levels were significantly increased in atrovastatin (Ator) treated mice (*P

Figure 5

(A) Representative semi-quantitative RT-PCR for tPA and PAI-1. mRNA levels for tPA were elevated in atorvastatin (Ator) treated mice compared to vehicle treate controls. However, no significant change was detected for PAI-1. (B) Densitometric analysis of tPA mRNA levels normalized to beta-actin levels (mean+SEM). tPA levels were significantly increased in atrovastatin (Ator) treated mice (*P

Figure 5

(A) Representative semi-quantitative RT-PCR for tPA and PAI-1. mRNA levels for tPA were elevated in atorvastatin (Ator) treated mice compared to vehicle treate controls. However, no significant change was detected for PAI-1. (B) Densitometric analysis of tPA mRNA levels normalized to beta-actin levels (mean+SEM). tPA levels were significantly increased in atrovastatin (Ator) treated mice (*P

Figure 6

24 hr ischemic lesion volumes (mean+SEM) in eNOS knockout mice after embolic focal ischemia were significantly reduced (*P