Korean Journal of Cerebrovascular Surgery 2006;8(2):102-106.
Published online June 1, 2006.
Difference in Induction Rate of Experimental Cerebral Aneurysm According to High Salt, High Lipid and Normal Diet.
Choi, Hwan Young , Yi, Jin Seok , Lee, Hyung Jin , Yang, Ji Ho , Lee, Il Woo
Department of Neurosurgery, School of Medicine, Pusan National University, Busan, Korea. chwachoi@pusan.ac.kr
Abstract
OBJECTIVE
An intracranial aneurysm is an important acquired cerebrovascular disease that can cause a catastrophic subarachnoid hemorrhage. Atherosclerosis is one of possible mechanism, but its contribution to aneurysm formation is unclear. Experimentally induced cerebral aneurysm rate by high lipid diet was evaluated and compared with high salt and normal diet to elucidate the role of lipid metabolism in the process of cerebral aneurysm formation. METHODS: Thirty-seven 7-week-old male Sprague-Dawley (SD) rats received a cerebral aneurysm induction procedure. The control animals (n=11) were fed a normal diet, and the experimental animals were fed a diet containing 8% salt (n=15) and high lipid (n=11) for three months. Three months after the operation, the rats were killed, their cerebral arteries were dissected, and the regions of the bifurcation of the right anterior cerebral artery-olfactory artery (ACA-OA) bifurcations were examined histologically and aneurysm induction rates among three groups were analysed. RESULTS: Average systolic blood pressures after 3 months feeding in three groups (high salt diet group, high lipid diet group and normal diet group) were 175.9+/-3.4 mmHg, 133.7+/-5.1 mmHg and 128+/-2.9 mmHg, respectively. The difference between high lipid group and normal diet group was not significant (P=0.215). The aneurysm induction rate in three group were 87%, 63% and 36%. The difference between high lipid diet group and normal diet group was significant (Pearson k2, P=0.029). CONCLUSIONS: High lipid diet significantly increase the cerebral aneurysm induction rate in experimentally induced cerebral aneurysm model of rats. That suggests a possible adverse role for hyperlipidemia leading to aneurysm formation. Further studies are necessary to elucidate the exact role of hyperlipidemia in the pathophysiology of cerebral aneurysm.
Key Words: Cerebral aneurysm, High lipid diet, Hyperlipidemia

Introduction


  
Subarachnoid hemorrhage (SAH) remains a serious disease despite attempts to improve outcome with medical and neurosurgical treatment. The overall mortality is still approximately 50%to 60%. So, many studies have been performed for finding etiology or affecting factors. Many factors have been reported and the two major risk factors identified are chronic hypertension and current smoking.22) Atherosclerosis is also thought to be one of them but its contribution to aneurysm formation is unclear. In some study, hyperlipidemia is not a risk factor for growth of aneurysm and instead a protective factor.10)22) High lipid diet induce atherosclerosis and make change in vascular integrity. Proteolytic enzymes in the complicated plaque may digest elastic fibers in the media, resulting in aneurysmal enlargement.9)23) The pathology is interpreted as atherosclerotic induced medial disruption of the aneurysmal wall and atrophic degeneration with fibrous replacement of elastin and inflammation of the media and adventitia.6) We designed the study for evaluating about influence of high lipid diet on aneurysm.
   High salt diet is a proven factor of aneurysm. This increases blood pressure and induces hypertension, so facilitates development of aneurysm. We added high salt diet group in this study.
   We devided rats into three groups and compared the average systolic blood pressure before operation and before death, and induction rate of cerebral aneurysm.

Materials and Methods

Induction of Experimental Cerebral Aneurysms
  
A total of 37 male, 7-week-old Sprague-Dawley rats each weighing 200 to 300 g were used in this study. The left common carotid artery and posterior branches of both renal arteries were ligated to induce cerebral aneurysms.1)4) 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 remaining 26 experimental animals were fed a diet containing 8% salt diet (n=15) and 42% lipid (TD96125; Harlan-Teklad, Madison, Wisc: n=11) for 3 months. Animal care and experiments complied with community standards on the care and use of laboratory animals.
   Systolic blood pressure was measured twice by the tail-cuff plethysmographic method with rats in an unanesthetized state just before the operation and just before death.

Tissue Preparation
  
Three months after the induction procedure, a total of 37 experimental rats were deeply anesthetized with ether and perfused transcardially with 0. 1 mol/L PBS followed by 4% paraformaldehyde at a pressure of approximate 200 mm H2O. Cerebral arteries were stripped from their brains 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. The each samples were embedded in OCT compound (Tissue-Tek), and 4-μ m thin sections were cut with a Leica CM 1850 and mounted on silane-coated slides. We could make 10 to 15 slides per 1 sample containing the aneurysm. In this study, we defined the terms to describe the development of cerebral aneurysms as our previous study1): Early cerebral aneurysm consists of discontinuity of the internal elastic lamina and no apparent outward bulging of the vascular wall. Advanced cerebral aneurysm refers to an obvious outward bulging of the arterial wall with complete disappearance of the internal elastic lamina (Fig. 1).
   Three independent researchers assessed the histopathological changes in a blinded manner.

Statistical Analysis
  
The values of blood pressure was xpressed as mean±SD. Statistical analysis in average systolic blood pressure after 3 months feeding were performed by using χ2 tests. Differences between each group were considered statistically significant at P<0.05. The valves of aneurysm induction rate was expressed as sum of percentage of early and advanced aneurysm rate. Statistical analysis in aneurysm induction rate among 3 groups were performed by using Pearson k2 tests.

Results

Blood Pressure Measurement
  
The average systolic blood pressure of high salt diet group just before operation was 102.5±3.6 mmHg, and that just before death was 175.9±3.4 mmHg. The average systolic blood pressure of high lipid diet group just before operation was 103.7±4.2 mmHg, and that just before death was 133.7±5.1 mmHg. The average systolic blood pressure of normal diet group just before operation was 102.8±2.3 mmHg, and that just before death was 128±2.9 mmHg. The difference of average systolic blood pressure after 3 months feeding between high salt diet group and high lipid diet group was significant (P<0.001). The difference between high salt diet group and normal diet group was also significant (P<0.001). But the difference between high lipid group and normal diet group was not significant (P=0.215)(Table 1).

Light Microscopic Study
  
In high salt diet group (n=15), the ACA-OA bifurcation showed no aneurysmal change in 2, early aneurysmal change in 3 (20%), and advanced aneurysmal change in 10 rats (67%). Aneurysm induction rate in high salt diet group was 87%. In high lipid diet group (n=11), no change in 4, early in 2 (18%), and advanced in 5 rats (45%). Aneurysm induction rate in high lipid diet group was 63%. In normal diet group (n=11), no change in 7, early in 0, and advanced in 4 rats (36%). Aneurysm induction rate in normal diet group was 36%.
   The difference of aneurysm induction rate between high lipid diet group and normal diet group was significant (Pearson k2, P=0.029)(Table 2).

Discussion

   The mechanisms of intracranial berry aneurysm 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.2)17)18)19) In particular, hemodynamic stress has been shown in many investigations to be the major cause of various degenerative changes in cerebral aneurysm formation.3)7)12)18)21) Genetic factors, hypertension, atherosclerosis, age, sex and smoking have also been reported to affect this disease.11)13)14)16) Atherosclerosis may be present, but its contribution to intracranial berry aneurysm formation is unclear.
   Chronic imflammatory responses contribute to the pathogenesis of atherosclerosis in humans and experimental models.8)15) Under hypercholesterolemic conditions, oxidized low density lipoprotein and other modified forms of lipid accumulate in the vessel intima. Inflammatory cells such as monocytes and T lymphocytes migrate through the endothelium, infiltrate the vessel wall, and release cytokines and growth factors, causing medial smooth muscle cells (SMC) to proliferate and migrate into the intima. As atherosclerotic plaque develops, the development of lumen stenosis and arterial enlargement may occur. This may compensate for increasing plaque size and prevent the development of luminal narrowing. This process may also occur as a result of local increase in flow associated with periodic temporary narrowing of the lumen produced by an encroaching intimal plaque. The increase in wall stress may be expected to stimulate endothelial release of nitric oxide or other factors, resulting in smooth muscle relaxation in the media and artery dilatation.24) Cause of aneurysmal atherosclerosis of human and experimental abdominal aorta remains unsettled. Although matrix metalloproteinases,20) genetic factors, and apoptosis,5) have been shown to play important roles in the development of abdominal aneurysms, the etiologic relationship is unclear. Clinical, experimental, and morphologic evidence supports the hypothesis that atherosclerotic lesion of the intima plays important role in the development of abdominal aneurysms. Plaque deposition associated with localized dilatation, thinning of the media, and loss of elastic lamellae may predispose that segment of aorta to subsequent abdominal aneurysm formation.23)
   In the previous study we showed marked positive immunoreactivity against apolipoprotein E in the medial SMC layer of early aneurysmal change.1) These findings may be interpreted that apolipoprotein E and lipid metabolism have a possible role in early events of SMC layer leading to aneurysm formation. But we don’t know exact role of apolipoprotein E in early aneurysmal change. The results of present study suggest that hyperlipidemia 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 bifurcation to subsequent cerebral aneurysm formation. Further studies should be aimed at specific hypotheses regarding the potential biological role of lipid metabolism and apolipoprotein E in aneurysm pathogenesis.

Conclusion

   High lipid diet significantly increase the cerebral aneurysm induction rate in experimentally induced cerebral aneurysm model of rats. That suggests a possible adverse role for hyperlipidemia leading to aneurysm formation. Further studies are necessary to elucidate the exact role of hyperlipidemia in the pathophysiology of cerebral aneurysm.


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