Peroxisome proliferator-activated receptor-alpha activation as a mechanism of preventive neuroprotection induced by chronic fenofibrate treatment

Abstract

The treatment of ischemic strokes is limited to the prevention of cerebrovascular risk factors and to the modulation of the coagulation cascade during the acute phase. A new therapeutic strategy could be to preventively protect the brain against noxious biological reactions induced by cerebral ischemia such as oxidative stress and inflammation to minimize their neurological consequences. Here, we show that a peroxisome proliferator-activated receptor (PPAR-alpha) activator, fenofibrate, protects against cerebral injury by anti-oxidant and anti-inflammatory mechanisms. A 14 d preventive treatment with fenofibrate reduces susceptibility to stroke in apolipoprotein E-deficient mice as well as decreases cerebral infarct volume in C57BL/6 wild-type mice. The neuroprotective effect of fenofibrate is completely absent in PPAR-alpha-deficient mice, suggesting that PPAR-alpha activation is involved as a mechanism of the protection against cerebral injury. Furthermore, this neuroprotective effect appears independently of any improvement in plasma lipids or glycemia and is associated with (1) an improvement in middle cerebral artery sensitivity to endothelium-dependent relaxation unrelated to an increase in nitric oxide synthase (NOS) type III expression, (2) a decrease in cerebral oxidative stress depending on the increase in numerous antioxidant enzyme activities, and (3) the prevention of ischemia-induced expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in cerebral vessels without any change in NOS II expression. These data demonstrate that PPAR-alpha could be a new pharmacological target to preventively reduce the deleterious neurological consequences of stroke in mice and suggest that PPAR-alpha activators could preventively decrease the severity of stroke in humans.

Figure 1.

PPAR-α activation as a mechanism of fenofibrate-induced preventive neuroprotection. In A, Apo E-deficient mice were fed a diet containing 0.2% fenofibrate or placebo (n = 5 per group) for 14 d before a 60 min MCA occlusion was performed. In these conditions, the increased susceptibility to cerebral ischemia of Apo E-deficient mice and the neuroprotective effect of fenofibrate appear only in the cortical area. ^{*, #}p < 0.05. B, Using the same procedure, cortical infarct volumes in C57BL/6 wild-type mice (n = 6 per group) are reduced by 46% after fenofibrate treatment when striatal infarct volumes remain similar. ^**p < 0.01. C, Preventive neuroprotective effect of fenofibrate is confirmed in SV129 wild-type mice (n = 5 per group) submitted to a 90 min MCA occlusion. Such a neuroprotective effect is not reproduced in PPAR-α-deficient mice (n = 4 per group), just as PPAR-α deficiency does not induce any increase in cerebral infarct volumes. ^**p < 0.01. NS, Not significant.

Figure 2.

Fenofibrate induces a slight improvement of MCA endothelial vasodilatation without any change in regional cerebral blood flow or NOS III expression. A, In mice treated with fenofibrate, there is no modification of regional cerebral blood flow (rCBF) as evaluated by laser Doppler (n = 5 in each group). NS, Not significant. B, NOS III is not overexpressed in brain of fenofibrate-treated mice. NS, Not significant. C, Application of low concentrations (3 × 10^-9 to 3 × 10^-8m) but not high concentrations (10^-7 to 3 × 10^-5m) of ACh on isolated MCA from rats treated with fenofibrate induces a slight improved sensitivity of endothelium-dependent relaxation as measured by the increase in artery diameter from 5-HT preconstricted level (n = 5 in each group). ^*p < 0.05.

Figure 3.

Chronic administration of fenofibrate modulates oxidative stress and antioxidant enzyme activities in the brain. A, B, Thiobarbituric acid-reactive substances (TBARS) were measured in the cortex (A) and striatum (B) of fenofibrate-treated Apo E-deficient mice and untreated C57BL/6 wild-type mice and then compared with the TBARS level in untreated Apo E-deficient mice (n = 5 per group). It appears that in Apo E-deficient mice, the basal level of TBARS is increased in the cortical area, whereas this oxidative stress marker is normalized by 14 d fenofibrate pretreatment. Moreover, there is no TBARS level change in the striatum. ^**p < 0.001; ^#p < 0.05. C, D, Antioxidant enzyme activities were measured in brain homogenates of C57BL/6 wild-type mice after a 14 d period under fenofibrate (n = 5) or placebo (n = 7). Four of the main antioxidant enzymes, copper/zinc superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reductase (GR), and glutathione S-transferase (GST), are significantly increased in fenofibrate-treated mice, whereas manganese SOD and catalase are not. ^*p < 0.05; ^**p < 0.01. E, Conversely to the measure of SOD activity, Western blot analysis does not show any difference in the expression of this enzyme in placebo and fenofibrate-treated animals as compared with control (EGF-stimulated A431 cells). Densitometric values from placebo and fenofibrate-treated animals were not different. NS, Not significant.

Figure 4.

Chronic administration of fenofibrate prevents ischemia-induced vascular expression of VCAM-1 and ICAM-1. VCAM-1 and ICAM-1 expression were evaluated in sham or mice subjected to a cerebral ischemia after a 14 d treatment period with or without fenofibrate. The intensity of staining was measured as a mean of five animals in each group. A, B, No expression is shown of VCAM-1 (A; cresyl violet counterstaining) or ICAM-1 (B; without counterstaining) in the brain of sham animals. C, D, VCAM-1 (C) and ICAM-1 (D) are induced after a 1 hr ischemia after a 24 hr reperfusion period in untreated animals in which such expression involves mainly vessels in the ischemic area. E, F, Adhesion molecule expression is prevented in ischemic mice receiving the fenofibrate treatment. Magnification: 20×. Scale bars, 50 μm.

Figure 5.

A 14 d fenofibrate treatment prevents ischemia-induced VCAM-1 and ICAM-1 but not NOS II overexpression. Western blot analysis was performed in brain homogenates of placebo and fenofibrate-treated mice and then compared with control animals injected with lipopolysaccharide (1.5 mg/kg, 24 hr before). A, B, Representative blots as well as densitometric values show a decrease in ischemia-induced VCAM-1 (A) and ICAM-1 (B). ^*p < 0.05; ^**p < 0.01. C, Conversely, ischemia-induced NOS II overexpression was not modified by fenofibrate pretreatement. NS, Not significant.