In this study, we demonstrated that the pleiotropic 3-hydroxyl-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor, atorvastatin, when administered prophylacticly, ameliorates early brain injury and cerebral vasospasm after experimental SAH. Inhibition of the caspase-dependent apoptotic pathways, while not other apoptosis-related proteins, was observed after treated with atorvastatin. The neuroprotective effects may be involved in its potential anti-apoptosis mechanisms. Administration of caspase inhibitors has been found to reduce caspase-3 activation after SAH [5, 10]. However, in the present research, atorvastatin, which is not a direct inhibitor of caspase, significantly reduced caspase-3 activation. It has been documented that P53 plays an orchestrating role in apoptotic cell death after experimental SAH . Expression of P53 and caspase-3, 8 was decreased by P53 inhibitor, Pifithrin-α, which showed that P53 acting as an upstream of caspase activation. However, expression of P53 and other apoptosis-related proteins were not influenced by atorvastatin based on our results. It can be speculate that, at least in the apoptosis pathway, atorvastatin has the direct effect on caspase-3. Pleiotropic effects of statins has been found after SAH, such as increased phosphorylation of Akt and endothelial nitric oxide synthase . However, whether anti-apoptotic effect of atorvastatin via these pathways was not determined in the present research.
Despite intense research efforts in aneurismal SAH, we currently possess a very limited understanding of the underlying mechanisms that result in brain injury . However, a number of studies have recently indicated that apoptosis may be a major player in the pathogenesis of secondary brain injury after SAH . As a result, the apoptotic cascades present a number of potential therapeutic opportunities that may ameliorate secondary brain injury. Experimental data suggest that these cascades occur very early after initial insult and may be related directly to physiologic sequela commonly associated with SAH . In accordance with the previous reports, our data imply that numbers of TUNEL positive cells are significantly increased in SAH rats, while atorvastatin administered ahead markedly inhibited apoptosis after SAH.
Apoptosis requires activation of caspases, a cascade of cysteine proteases activated by proteolytic cleavage, which target key homeostatic and structural proteins, such as actin and fodrin as well as nuclear proteins like poly (ADP-ribose) polymerase (PARP), leading to DNA fragmentation and cell death. Caspase-3 activation is considered one of the final steps responsible for apoptosis . Caspase-3 can proteolytically cleave several crucial cellular proteins inducing the characteristic changes associated with apoptosis including PARP-1, considered a hallmark biochemical characteristic of apoptosis. PARP-1 cleavage facilitates DNA alteration and nuclear disassembly and ensures the completion of the energy-dependent apoptotic process. In this study, atorvastatin significantly reduces the increased caspase-3 protein expression observed 24 hour after the insult. Although several studies have shown that statins may cause apoptosis in different cell lines, including neuronal cells [14, 15], our data show that administration of atorvastatin to rats reduces the caspase-dependent apoptotic signal induced by SAH. In agreement with our results, it has been recently reported that another statin, pravastatin, reduces apoptosis in the hippocampus of adult rats after transient forebrain ischemia . The reason for the different effect observed after "in vitro" or "in vivo" exposure to statins is unclear. One possibility is that statins do not exert a proapoptotic effect on brain cells "in vivo". This hypothesis is supported by the observation that caspase-3 activation and PARP cleavage were reduced in simvastatin-treated control animals or in the contralateral side of simvastatin-treated ischemic animals . Another possibility is that the pro-apoptotic effect on inflammatory cells and the inhibition of leucocyte function induced by statins perhaps overcomes the potential pro-apoptotic effect they might have on neuronal and/or glial cells and the ultimate result is neuroprotection. Hydroxymethylglutaryl coenzyme A reductase inhibitors have an attractive profile for cerebrovascular research. Statins have been demonstrated to reduce stroke events independent of their lipid-lowering effect as well as to reduce mortality significantly after myocardial infarction . We report here that prophylactic treatment with atorvastatin reduces caspase-3 and caspase-8 expression suggesting that its neuroprotective effect may result from a reduced number of neurons undergoing apoptotic cell death. To date, the apoptotic pathways believed to play a major role in SAH-induced apoptosis are the death receptor and TNF-a, p53, and the caspase dependent cascades . However, to the best of our knowledge, multiple level inhibitors have not been used. An example of this might be to selectively block the limiting steps of the caspase independent and -dependent cascades and the bcl-2 family proteins. But in our present study, protein levels of bcl-2 and bax expression did not differ statistically among the three groups, suggesting that bcl-2 pathway may not participate into the apoptosis involved in experimental SAH. As to p53, AIF, and Cytochrome C, our data indicated an upregulation of these apoptosis-related proteins after SAH. However, it was not affected by atorvastatin, the underlying mechanisms need further investigation. P53 was thinked as a key molecular mediated apoptosis after SAH. Inhibition of P53 with PFTα down-regulated expression of other apoptosis-related proteins, including caspase-3 and caspase-8 . In this research, P53 was not inhibited obviously by atorvastatin. This suggested that a unique mechanism of apoptosis inhibition underlying in atorvastatin, which made it more safety for treating SAH than other multi-levels apoptosis inhibitors.
Effects of statins on cerebral vasospasm and neurological outcome after SAH were still controversial [20, 21]. Ameliorating cerebral vasospasm, improving cerebral autoregulation, and reducing vasospasm-related DID of statins were observed in some researches . But based on the more recent report, statins using was not associated with less vasospasm and improved outcome after SAH . Difference in enrolled patients, research methods, and outcome measurement may explain the contradictions. The contradictions highlighted the importance of basic study and clinical trials for statins after SAH. As apoptosis had been observed in hippocampus, blood-brain barrier (BBB), and vasculature in SAH patients , apoptosis inhibition proved by the present research may be partly elucidate the effects of long-term neurological improvement of statins. Based on the current research, statins may be beneficial for SAH treatment due to its effects of ameliorating cerebral vasospasm and improving neurological outcome.