Korean Journal of Cerebrovascular Surgery 2010;12(3):196-201.
Published online September 1, 2010.
Effects of Atorvastatin on the Induction of Experimental Cerebral Aneurysm in a High Lipid Diet Rat Model.
Lim, Jeong Kyu , Yeo, In Sung , Yi, Jin Seok , Lee, Hyung Jin , Yang, Ji Ho , Lee, Il Woo
Department of Neurosurgery, Daejeon St Mary's Hospital, College of Medicine, The Catholic University of Korea, Daejeon, Korea. yangjiho@catholic.ac.kr
Abstract
OBJECTIVE
Previously, we reported that a high lipid diet significantly increases the induction rate of cerebral aneurysm (CA) formation in an experimentally induced CA rat model, suggesting that hypercholesterolemia with chronic inflammation leads to aneurysm formation. To elucidate the role of hypercholesterolemia in CA formation, experimentally induced CA was evaluated in rats fed a high lipid diet and treated with low and high doses of atorvastatin. METHODS: Thirty-seven, 7-week-old male Sprague-Dawley rats underwent a CA induction procedure. The control animals (n = 11) were fed a normal diet, and the experimental animals (n = 26) were fed a diet containing high lipid content for 3 months. The experimental group comprised a high-dose atorvastatin group (20 mg/kg/day, n = 15) and low-dose atorvastatin group (1 mg/kg/day, n = 11). Three months after the operation, induction of CA formation in the three groups was analyzed. RESULTS: Induced CA formation was 67%, 63%, and 36% in the control, high lipid/high atovastatin, and high lipid/low atovastatin group, respectively. The differences resulting from high-dose and low-dose atorvastatin were significant (Pearson k2, P = 0.028 and 0.029, respectively). CONCLUSIONS: A high lipid diet can significantly increase induction of CA formation. However, the lack of decreased induction in atorvastatin-treated animals suggests that high and low doses of atorvastatin do not inhibit the potential effects of hypercholesterolemia on CA formation. Further studies, such as those utilizing apolipoprotein E knockout mice, are necessary to elucidate the exact role of hypercholesterolemia in the pathophysiology of CA.
Key Words: Cerebral aneurysm, High lipid diet, Hypercholesterolemia, Atorvastatin
 

Introduction


The pathophysiology of cerebral aneurysm (CA) formation and progression is closely linked to vascular inflammation. Various inflammatory cells, especially macrophages, accumulate in the aneurysmal walls of human and experimentally induced CA in rats.2) Induction of experimental CA in rats without performing any direct manipulations of the cerebral artery itself has been reported. 5) The experimental CA in the rat model resembles the human version in the anatomic location and histological structure.11)  This model enables the exploration of the process of CA development.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are widely used lipid-lowering drugs. In addition, statins have vascular protective effects and increase nitric oxide bioavailability by the upregulation and activation of endothelial nitric oxide synthase.12) Statins also inhibit the expression of several matrix metalloproteinases (MMPs) in smooth muscle cells and macrophages.14)

In this study, a procedure to induce CA in rats was developed. The procedure involved the use of a high lipid diet, after which animals were treated with statins. The purpose of this study was to determine whether treatment with statins would have a favorable effect on the induction of experimental CA. To address this question, we examined the effects of atorvastatin (Lipitor, Pfizer), one of the most commonly used HMG-CoA reductase inhibitors in clinical practice, on CA formation in this rat model.


Materials and Methods


1.  Induction of Experimental CAs

Thirty-seven, 7-week-old Sprague-Dawley rats weighing 200~300 g were used. The left common carotid artery and posterior branches of both renal arteries were ligated to induce CA formation. These procedures were performed under intraperitoneal sodium pentobarbital anesthesia (40 mg/kg) with additional injections when necessary. After the operation, the 11 control animals were fed a normal diet, and the remaining 26 experimental animals were fed a diet consisting of 42% lipid (TD96125; Harlan-Teklad, Madison, WI: n = 26) for 3 months. The high lipid group was divided into high-dose atorvastatin (20 mg/kg/day, n = 15) and low dose atorvastatin (1 mg/kg/day, n = 11) groups. Atorvastatin was orally administered using a syringe. Animal care and experiments complied with community standards on the care and use of laboratory animals. Systolic blood pressure was measured by the tail-cuff plethysmographic method with rats in an unanesthetized state just before the operation and immediately before death.


2.  Tissue Preparation

Three months after the induction procedure, the 37 rats were deeply anesthetized with ether and perfused transcardially with 0.1 mol/L phosphate buffered saline (PBS) followed by 4% paraformaldehyde at a pressure of approximately 200 mmH2O. Cerebral arteries were stripped from the brains of rats under a surgical microscope. The right anterior cerebral artery-olfactory artery (ACA-OA) bifurcations, where aneurysmal changes of various degrees were speculated to be induced, were cut from the circles of all rats. Each sample was embedded in OCT compound (Tissue-Tek), and 4 μm-thick sections were cut with a Leica CM 1850 and mounted onto silane-coated slides. Ten-to-fifteen slides could be prepared from each aneurysm sample. The histological features of early aneurysmal changes were thinning of the medial smooth muscle layer and fragmentation of internal elastic lamina, but with no apparent outward bulging of the vascular wall. In the advanced aneurysm group, wall dilatation became apparent. In proportion to the dilatation size, the wall tended to become thinner. In the thinner parts of the wall, the number of smooth muscle cells decreased and cell disarrangement increased. The degenerative changes of the internal elastic lamina tended to be advanced in proportion to the changes of the medial layer. The internal elastic lamina was discontinuous near the entrance of the lesion and nearly disappeared at the dome. No aneurysmal changes were observed in the left ACA-OA bifurcations of any rat. The findings of the walls were similar to those of the normal arterial wall, although they tended to be thicker. Three independent researchers assessed the histopathological changes in a blinded manner.


3.  Double Staining

The sections were washed three times with 0.1 mol/L PBS for 5 minutes each. After a section had been blocked with 5% normal donkey serum, double staining was performed using goat anti-mouse apolipoprotein E (ApoE) antibody (Santa Cruz Biotechnology) and mouse anti-macrophage antibody (Chemicon) as primary antibodies. The sections were incubated with the solution of primary antibodies overnight at 4℃. The slides were then washed three times with PBS and subsequently incubated with secondary antibodies (anti-goat Alexa-Fluor 488 antibody and anti-mouse Alexa-Fluor 594 antibody; Molecular Probes) solution for 1 hour at room temperature. After they were washed three times with PBS, the sections were examined as described above (Fig. 1).


4.  Statistical Analysis

The values of blood pressure are expressed as means± SD. Statistical analysis in average systolic blood pressure after 3 months of feeding were performed using χ2 tests. Differences between groups were considered statistically significant at P < 0.05. The values of aneurysm induction rates were expressed as the sum of the percentages of early and advanced aneurysm rates. Statistical analysis of the aneurysm induction rate among the three groups was performed using Pearson k2 tests.

Results


1.  Blood Pressure Measurement

The average systolic blood pressure in the high-dose atorvastatin group just before the operation was 103.1 ± 2.9 mmHg, and the pressure just before death was 135.5 ± 3.7 mmHg. The average systolic blood pressure in the low-dose atorvastatin group just before the operation was 102.9 ± 3.8 mmHg, which increased to 132.5 ± 7.8 mmHg immediately before death. The average systolic blood pressure in the normal diet group just before the operation was 102.6 ± 5.4 mmHg, the pressure immediately before death was 126.3 ± 7.7 mmHg. The differences in average systolic blood pressure after 3 months of feeding between the high-dose atorvastatin group and normal diet group, low-dose atorvastatin group and normal diet group, and the high- and low-dose atorvastatin groups were not significant (P = 0.235, 0.256, and 0.215, respectively; Table 1).


2.  Light Microscopic Study

In the high-dose atorvastatin group (n = 15), ACA-OA bifurcation resulted in no aneurysmal change, early aneurysmal change, and advanced aneurysmal change in five (33%), three (20%), and seven (47%) rats, respectively, resulting in an aneurysm induction rate of 67%. In the low-dose atorvastatin group (n = 11), no change, early change, and advanced change occurred in four (37%), three (27%), and four (36%) rats, respectively, resulting in an aneurysm induction rate of 63%. In the normal diet group (n = 11), no change, early change, and advanced change occurred in seven (64%), one (9%), and three (27%) rats, respectively, resulting in an aneurysm induction rate of 36%. Differences between the high-dose atorvastatin group, normal diet group and the low-dose atorvastatin group and normal diet group were significant (Pearson k2, P = 0.028 and 0.029, respectively; Table 2). 


Discussion


Atorvastatin (Lipitor, Pfizer), is a member of the drug class known as statins, which are used for lowering blood cholesterol. It also stabilizes plaques and prevents strokes through anti-inflammatory and other mechanisms. Similar to other statins, atorvastatin is a competitive inhibitor of HMG-CoA reductase, the rate-determining enzyme located in hepatic tissue that produces mevalonate, a small precursor molecule in the synthesis of cholesterol and other mevalonate derivatives. HMG-CoA reductase catalyzes the reduction of HMG-CoA to mevalonate, which is the rate-limiting step in hepatic cholesterol biosynthesis. Inhibition of this enzyme decreases de novo cholesterol synthesis, resulting in increased expression of low-density lipoprotein (LDL) receptors on hepatocytes. The increased LDL uptake by hepatocytes decreases the amount of LDL cholesterol in the blood. Similar to other statins, atorvastatin also reduces blood levels of triglycerides and slightly increases levels of high-density lipoprotein cholesterol.12)

The mechanisms of CA formation, growth, and rupture are complex. Inherent structural weaknesses of cerebral vessels, including the absence of an external elastic lamina and a unique branching pattern, together with pulsatile hemodynamic bombardment, lead to increase shear stresses at bifurcations.8) In particular, hemodynamic stress has been shown in many investigations to be the major cause of various degenerative changes during CA formation.11) In the case of abdominal aortic aneurysms (AAA), aneurysma have a definite relationship with atherosclerosis and chronic inflammation of arterial walls.6)9)12-15) A well-established animal model for hypercholesterolemia and arteriosclerosis is the ApoE knockout (KO) mice model.16) Widely used AAA models are induced in ApoE KO mice because arteriosclerotic changes in the arterial walls are a critical pathological feature of AAA.14) Clinical, experimental, and morphologic evidence supports the hypothesis that atherosclerotic lesion of the intima plays an important role in the development of abdominal aneurysms. Nevertheless, its contribution to CA formation is unclear. 

In a previous study, we showed marked positive immunoreactivity against ApoE in the medial SMC layer during early aneurysmal change.4) These findings suggested that ApoE and lipid metabolism have a possible role in early events in the SMC layer that led to aneurysm formation. In another study, we reported that a high lipid diet significantly increases the induction rate of experimentally induced CA in rats.3) That provided evidence of a possible adverse role for hypercholesterolemia in aneurysm formation. The results of the present study support the view that a hypercholesterolemic state will promote lipid accumulation in the vessel intima and early atherosclerotic plaque change. Plaque deposition associated with localized hemodynamic stress and dilatation, thinning of the media, and loss of elastic lamellae may predispose that segment of the bifurcation to subsequent CA formation.

In studies using simvastatin, the progression of experimentally induced CA in rats1)and AAA in mice7)14) was suppressed. In a study by Akoi and colleagues,1) a high-salt diet was used instead of a high-lipid diet, and the investigators focused on progression of CA. Simvastatin treatment prevented thinning of aneurysmal walls of preexisting CAs. The authors suggested that simvastatin not only inhibits degenerative changes in vascular walls but also promotes the repair process. Enlargement of preexisting CAs was also inhibited by simvastatin. However, presently, a high-lipid diet was used and the focus was on the CA induction rate. Atorvastatin at low and high doses failed to decrease this induction rate. These results suggested that different mechanisms might be involved in CA formation compared with AAA formation. Histopathological studies of CAs have revealed no apparent evidence of arteriosclerotic changes in aneurysm walls. Moreover, CAs usually develope at the bifurcation site of arteries in both experimental animals and humans. Compared with CAs, AAAs form at the sidewall of the aorta and not at the arterial bifurcation. In cases of CA, hemodynamic stress and hypertension are serious risk factors. However, hypercholesterolemia is not a serious risk factor for CA formation.10) Statin treatment may suppress the progression of CAs but not decrease their induction.


Conclusion


A high lipid diet significantly increased the induction rate of experimental CA in a rat model. However, atorvastatin-treated groups did not show decreases in induction rates, suggesting that atorvastatin does not inhibit the potential adverse role of hypercholesterolemia in CA formation. Further studies, such as those in ApoE KO mice, are necessary to elucidate the exact role of hypercholesterolemia in the pathophysiology of CAs.


References

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  2)    Aoki T, Kataoka H, Moriwaki T, Nozaki K, Hashimoto N: Role of TIMP-1 and TIMP-2 in the progression of cerebral aneurysm. Stroke 38:2337-2345, 2007

  3)    Choi HW, Yi JS, Lee HJ, Yang JH, Lee IW: Difference in induction rate of experimental cerebral aneurysm according to high salt, high lipid and normal diet. Korean J CerebrovasculSurg 8:102-106, 2006

  4)    Choi YM, Yi JS, Lee HJ, Yang JH, Lee IW: Apolipoprotein E expression in experimentally induced intracranial aneurysms of the rats. J Korean NeurosurgSoc 39:46-51, 2006

  5)    Hashimoto N, Handa H, Hazama F: Experimentally induced cerebral aneurysms in rats. Surg Neurol 10:3-8, 1978

  6)    Holmes DR, Lopez-Candales A, Liao S, Thompson RW: Smooth muscle cell apoptosis and p53 expression in human abdominal aortic aneurysms. Ann NY Acad Sci 800:286-287, 1996

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  8)    Kondo S, Hashimoto N, Kikuchi H, Hazama F, Nagata I, Kataoka H: Apoptosis of medial smooth muscle cells in the development of saccular cerebral aneurysms in rats. Stroke 29:181-188, 1998

  9)    Libby P: Inflammation in atherosclerosis. Nature 420:868-874, 2002

10)    Miyazawa N, Akiyama I , Yamagata Z: Risk factors for growth of unruptured intracranial aneurysms: follow-up study by serial 0.5-T magnetic resonance angiography. Neurosurg 58:1047-1053, 2006

11)    Nakatani H, Hashimoto N, Kang Y, Yamazoe N, Kikuchi H, Yamaguchi S et al: Cerebral blood flow patterns at major vessel bifurcations and aneurysms in rats. J Neurosurg 74:258-262, 1991

12)    Nawrocki JW, Weiss SR, Davidson MH, Sprecher DL, Schwartz SL, Lupien PJ et al: Reduction of LDL cholesterol by 25% to 60% in patients with primary hypercholesterolemia by atorvastatin, a new HMG-CoA reductase inhibitor. Arterioscler Thromb Vasc Biol. 15:678-682, 1995

13)    Ross R: The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature (London) 362:801-809, 1993

14)    Steinmetz EF, Buckley C, Shames ML, Ennis TL, Vanvickle-Chavez SJ, Mao D et al: Treatment with simvastatin suppresses the development of experimental abdominal aortic aneurysms in normal and hypercholesterolemic mice Ann Surg 241:92-101, 2005

15)    Thompson RW, Parks WC: Role of matrix metalloproteinases in abdominal aortic aneurysms. Ann NY Acad Sci 800:157-174, 1996

16)    Zhang SH, Reddick RL, Piedrahita JA Maeda N: Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science 258:468-471, 1992



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