Endothelial injury and inflammatory response induced by hemodynamic changes preceding intracranial aneurysm formation: experimental study in rats

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Object

Intracranial aneurysms are the leading cause of subarachnoid hemorrhage, which is associated with high morbidity and mortality rates. Despite advances in the microsurgical and endovascular treatment of intracranial aneurysms, little is known about the mechanisms by which they originate, grow, and rupture. To clarify the series of early events leading to formation of intracranial aneurysms, the authors compared aneurysmal morphological changes on vascular corrosion casts with parallel pathological changes in the cerebral arteries of rats.

Methods

The authors induced cerebral aneurysms by renal hypertension and right common carotid artery ligation in 40 male Sprague–Dawley rats; 10 intact rats served as the controls. The anterior cerebral artery–olfactory artery bifurcation was assessed morphologically by using vascular corrosion casts of Batson plastic reagent and immunohis-tochemically by using antibodies against endothelial nitric oxide synthase, α–smooth muscle actin, macrophages, and matrix metalloproteinase–9.

Results

Surgically treated rats manifested different degrees of aneurysmal changes. Based on these staged changes, the authors propose that the formation of intracranial aneurysms starts with endothelial injury at the apical intimal pad (Stage I); this leads to the formation of an inflammatory zone (Stage II), followed by a partial tear or defect in the inflammatory zone. Expansion of this defect forms the nidus of the intracranial aneurysm (Stage III).

Conclusions

This is the first study to demonstrate the in vivo mechanisms of intracranial aneurysm formation. The inflammatory response that follows endothelial injury is the basic step in the pathogenesis of these lesions. In this study the investigators have expanded the understanding of the origin of intracranial aneurysms and have contributed to the further development of measures to prevent and treat aneurysms.

Abbreviations used in this paper:ACA–OlfA = anterior cerebral artery–olfactory artery; α-SMA = α–smooth muscle actin; CCA = common carotid artery; eNOS = endothelial nitric oxide synthase; MMP-9 = matrix metalloproteinase–9; PBS = phosphate-buffered saline; SAH = subarachnoid hemorrhage; SMC = smooth-muscle cell.

Article Information

Address reprint requests to: Mohammad A. Jamous, M.D., Ph.D., Department of Neurosurgery, School of Medicine, The University of Tokushima, 3-18-15 Kuramotocho, Tokushima City, Tokushima 770-8503, Japan. email: mojamous@yahoo.com.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Bar graphs showing the mean systolic blood pressure (left) and CCA blood flow (right) in the control and experimental (operated) rats. Data represent means ± standard deviations (*p < 0.05).

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    Scanning electron microscopy studies of vascular corrosion casts of the ACA–OlfA bifurcation (A–E) and double immunostaining of the vascular wall (F–J; green staining shows eNOS and red shows α-SMA). A and F: Normal. A: Normal endothelial cells are seen at the apical intimal pad, and the endothelial cell markings are elongated in the direction of the blood flow. F: The endothelial and the smooth-muscle layer form two continuous layers. B and G: Stage I. B: Roughened apical intimal pad with irregularly shaped imprints (arrow). G: Loss of eNOS expression at the apical intimal pad (arrow). C and H: Stage IIA. C: Shallow elevation (arrow) surrounded by an area of depression of the apical intimal pad (arrowhead). H: Swelling of the vessel wall at the apical intimal pad; part of this swollen area lacks eNOS expression and shows subendothelial expression of α-SMA–positive cells (arrow). D and I: Stage IIB. D: Pyramid-shaped elevation of the apical intimal pad; note that the surface of this elevation is covered by abnormal imprints. I: Thinning and degradation of the smooth-muscle layer creates a defect in the inflammatory zone (arrow) and produces vessel wall protrusion. E and J: Stage III. E: Saccular aneurysm covered with abnormal imprints. J: Expansion of the inflammatory zone defect, and destruction and protrusion of the vessel wall representing the nidus of the cerebral aneurysm. OA = OlfA. Bars = 50 μm.

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    Early inflammatory changes characterized by SMC migration and macrophage infiltration (Stage IIA). A and B: Sagittal cut of the left ACA–OlfA bifurcation viewed at low (A) and high magnification (B) showing swelling of the apical intimal pad. C: Double immunostaining of an ACA–OlfA section with eNOS antibodies (green) and α-SMA (red) shows swelling of the vessel wall at the apical intimal pad; part of this swollen area lacks eNOS expression and shows migration of α-SMA–positive cells into the neointima (arrow). D: Triple immunostaining of an ACA–OlfA section with antibodies against eNOS (green), α-SMA (red), and macrophages (blue). Macrophage expression confirms the presence of an inflammatory zone. Bars = 50 μm.

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    Late inflammatory changes characterized by progression of inflammation, resulting in a defect within the inflammatory zone. A: Double immunostaining of an ACA–OlfA section. The eNOS antibodies (green) and α-SMA (red) demonstrate the thinning and destruction of the smooth-muscle layer. This creates a defect (arrow) in the inflammatory zone that appears to lack eNOS expression and has a thin layer of α-SMA–positive cells. B: Magnified view of panel A demonstrating the inflammatory zone defect (arrow). This defect lacks eNOS expression. C: Positive expression of macrophages (blue) around the inflammatory defect. D: Merged images. Lu = lumen. Bars = 10 μm.

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    Expansion of the inflammatory zone resulting in saccular aneurysm formation. A: Sagittal cut of the left ACA–OlfA bifurcation showing the nidus of a cerebral aneurysm (arrow). B: Double immunofluorescence labeling with antibodies against eNOS (green) and α-SMA (red) shows the lack of eNOS expression (arrows) in the aneurysm wall. Note the subendothelial migration of SMCs (arrowheads). The aneurysm cavity is limited to the area of SMC migration. C: Positive expression of MMP-9 (blue) resulting in vessel wall destruction. D: Merged images. Bars = 10 μm.

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    Schema outlining our hypothesis for cerebral aneurysm formation.

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