Critical roles of macrophages in the formation of intracranial aneurysm

Abstract

Background and purpose: abnormal vascular remodeling triggered by hemodynamic stresses and inflammation is believed to be a key process in the pathophysiology of intracranial aneurysms. Numerous studies have shown infiltration of inflammatory cells, especially macrophages, into intracranial aneurysmal walls in humans. Using a mouse model of intracranial aneurysms, we tested whether macrophages play critical roles in the formation of intracranial aneurysms.

Methods: intracranial aneurysms were induced in adult male mice using a combination of a single injection of elastase into the cerebrospinal fluid and angiotensin II-induced hypertension. Aneurysm formation was assessed 3 weeks later. Roles of macrophages were assessed using clodronate liposome-induced macrophage depletion. In addition, the incidence of aneurysms was assessed in mice lacking monocyte chemotactic protein-1 (CCL2) and mice lacking matrix metalloproteinase-12 (macrophage elastase).

Results: intracranial aneurysms in this model showed leukocyte infiltration into the aneurysmal wall, the majority of the leukocytes being macrophages. Mice with macrophage depletion had a significantly reduced incidence of aneurysms compared with control mice (1 of 10 versus 6 of 10; P<0.05). Similarly, there was a reduced incidence of aneurysms in mice lacking monocyte chemotactic protein-1 compared with the incidence of aneurysms in wild-type mice (2 of 10 versus 14 of 20, P<0.05). There was no difference in the incidence of aneurysms between mice lacking matrix metalloproteinase-12 and wild-type mice.

Conclusions: these data suggest critical roles of macrophages and proper macrophage functions in the formation of intracranial aneurysms in this model.

Figure 1. Representative intracranial aneurysms in hypertensive mice that received a single injection of elastase into the cerebrospinal fluid

Arrows indicate aneurysms. Large aneurysm formation was found along the Circle of Willis or its major branches (A-D). Dissection of aneurysms revealed saccular shape of the aneurysms (A, B). Some of the mice had multiple aneurysms (D). Bar = 1 mm, ACA: anterior cerebral artery, MCA: middle cerebral artery, PCA: posterior cerebral artery, ICA: internal carotid artery.

Figure 2. H&E, pan-leukocyte, and macrophage staining

A. A cerebral artery from a mouse in the sham operation group showed an endothelial cell lining with a thin layer of smooth muscle cells. B. Intracranial aneurysms had a partially thickened vascular wall with inflammatory-like cell infiltration. Endothelial cell layers seemed to be generally intact, but smooth muscle cell layers had thickened in the area with intense inflammatory cell infiltration. C, E. Cerebral artery from a mouse in the sham operation group showed a lack of inflammatory cells. D, G. Intracranial aneurysms had numerous leukocytes. Distribution of macrophages was similar to that of leukocytes. F. Double staining with anti-CD68 and anti-CD45 revealed that a majority of leukocytes in intracranial aneurysms in this model were macrophages.

Figure 3. Macrophage depletion and intracranial aneurysm formation

A. Mice with macrophage depletion treatment using clodronate liposome had a reduced incidence of intracranial aneurysms compared to mice that received PBS liposome (P < 0.05). B-G. While there were an abundant number of macrophages in the intracranial aneurysms from PBS liposome treated mice, middle cerebral arteries from mice in either the sham operation group or the macrophage depletion group did not show macrophage infiltration into the vascular wall. H. The number of macrophages was higher in mice treated with elastase, angiotensin-II, and PBS liposome compared to mice in the sham group or macrophage depletion group (n = 5 in each group, P < 0.05).

Figure 4

A. Macrophage staining of the spleen from mice that received clodronate liposome. Brown area indicates macrophage positive area. B. Clodronate liposome treatment decreased CD68 positive area in the spleen by 88% from the baseline (P < 0.05), showing an effective macrophage / monocyte reduction by the clodronate treatment. At two weeks, CD68 positive area returned to the baseline. C. Successful induction of hypertension by angiotensin-II.

Figure 5

A. MCP-1 knockout mice had a lower incidence of aneurysms compared to wild-type mice (P < 0.05). There was no difference in the incidence of intracranial aneurysms between MMP-12 knockout mice and wild-type mice. B-D. While macrophage infiltration was observed in the cerebral arteries of wild-type mice (B) and MMP-12 knockout mice (D), cerebral arteries in MCP-1 knockout mice showed a lack of macrophage infiltration (C). F. Successful induction of hypertension by angiotensin-II.