Elastase-induced intracranial aneurysms in hypertensive mice - PubMed (original) (raw)

Elastase-induced intracranial aneurysms in hypertensive mice

Yoshitsugu Nuki et al. Hypertension. 2009 Dec.

Abstract

Mechanisms of formation and growth of intracranial aneurysms are poorly understood. To investigate the pathophysiology of intracranial aneurysms, an animal model of intracranial aneurysm yielding a high incidence of large aneurysm formation within a short incubation period is needed. We combined two well-known clinical factors associated with human intracranial aneurysms, hypertension and the degeneration of elastic lamina, to induce intracranial aneurysm formation in mice. Roles of matrix metalloproteinases (MMPs) in this model were investigated using doxycycline, a broad-spectrum MMP inhibitor, and MMP knockout mice. Hypertension was induced by continuous infusion of angiotensin II for 2 weeks. The disruption of elastic lamina was achieved by a single stereotaxic injection of elastase into the cerebrospinal fluid at the right basal cistern. A total of 77% of the mice that received 35 milliunits of elastase and 1000 ng/kg per minute of angiotensin II developed intracranial aneurysms in 2 weeks. There were dose-dependent effects of elastase and angiotensin II on the incidence of aneurysms. Histologically, intracranial aneurysms observed in this model closely resembled human intracranial aneurysms. Doxycycline, a broad-spectrum MMP inhibitor, reduced the incidence of aneurysm to 10%. MMP-9 knockout mice, but not MMP-2 knockout mice, had reduced the incidence of intracranial aneurysms. In summary, a stereotaxic injection of elastase into the basal cistern in hypertensive mice resulted in intracranial aneurysms that closely resembled human intracranial aneurysms. The intracranial aneurysm formation in this model appeared to depend on MMP activation.

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Conflict of interest statement

Conflict of interest

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Figures

Figure 1

Figure 1. Representative intracranial aneurysms

A: a schematic view of normal cerebral arteries. B: Normal cerebral arteries including the Circle of Willis and its major branches are stained in blue. C–E: Base of the brain with intracranial aneurysm formations. Black arrows indicate intracranial aneurysm formations. Large intracranial aneurysm formations were found mostly along the right half of the Circle of Willis and its major branches. C: intracranial aneurysm at the right posterior communicating artery. D: intracranial aneurysm at the right middle cerebral artery. E: A mouse that died at day 12 had fresh blood clots along the right middle cerebral artery, revealing subarachnoid hemorrhage. Inside the blood clots of subarachnoid hemorrhage, there was a larger aneurysm formation. F: locations of intracranial aneurysms. Left panel shows a diagram of the cerebral arteries in mice, and right panel shows locations of aneurysms found in the mice that received 35 milli-units of elastase and 1000 ng/kg/min of angiotensin-II (n = 44). Majority of the intracranial aneurysms were located ipsilaterally to the elastase injection site. Small numbers of intracranial aneurysms were found on the contra-lateral side of the injection site as well as on the branches of the basilar artery. ACA: anterior cerebral artery, MCA: middle cerebral artery, ICA: internal carotid artery, PCA: posterior cerebral artery, SCA: superior cerebellar artery.

Figure 2

Figure 2. Dose-dependent effects of angiotensin-II and elastase on the incidence of intracranial aneurysms

A: Dose-dependent relationship between the incidence of intracranial aneurysms and the concentration of elastase. B: dose-dependent relationship between the incidence of aneurysms and the concentration of angiotensin-II. C: Systolic blood pressure in mice that received different doses of angiotensin-II. There was a dose-dependent relationship between angiotensin-II and systolic blood pressure. *: P < 0.05 compared to the group that received phosphate buffered saline (PBS) infusion instead of angiotensin-II. #: P < 0.05 compared to the baseline value in each group

Figure 3

Figure 3. Histological assessment of intracranial aneurysms in this model

In the normal cerebral artery from control mouse (A, C), there were two to three layers of smooth muscle cells (A2) and a single, thin continuous layer of endothelial cells (A3). Fibroblasts were very scarce (A4). Elastica van Gieson and trichrome (C1, C2) stainings showed one layer of elastic lamina. In an intracranial aneurysm (B, D), the thin vascular wall showed intact endothelial and smooth muscle cell layers, while the thick vascular wall showed discontinued endothelial cell layers and scattered, faint, smooth muscle staining (B2, B3). Elastica van Gieson and trichrome stainings revealed severely disorganized elastic lamina in both thin and thick portions of the artery (D, E). Bar = 100μm.

Figure 4

Figure 4. Panleukocyte (CD45), macrophage (CD68), and T-lymphocyte (CD4) stainings for a normal cerebral artery from the control mouse (A) and a representative intracranial aneurysm (B)

A normal basilar artery from the control mouse (A1, A2, A3)) showed absence of inflammatory cells. In the intracranial aneurysm tissue (B), leukocytes (CD45 positive cells) were present throughout the vascular wall (B1, B4). Distribution of macrophages (CD68 positive cells) generally overlapped with the leukocyte distribution (B2, B5). Small numbers of CD4 positive T-lymphocytes were present in the thin wall portion of the aneurysm. However, CD4 positive T-lymphocytes were not detected in the thick aneurysm wall (B3, B6). Bar = 100μm.

Figure 5

Figure 5. Roles of MMPs in intracranial aneurysm formation

In situ zymography with or without an MMP inhibitor was performed on a normal cerebral artery (B) and a representative intracranial aneurysm in this model (A). While the normal brain vasculature lacked any appreciable gelatinase activity (B), intracranial aneurysms showed intense fluorescence, indicating robust gelatinase activity (A). Pretreatment of the tissues with 1,10-phenanthroline monohydrate (MMP inhibitor) abolished the gelatinase activity (C), showing that the gelatinase activity observed in the intracranial aneurysm tissues was from MMPs. Incidence of intracranial aneurysms in four groups of mice is presented in G. The incidence of intracranial aneurysms in the control group (wild type) was 70%, consistent with the results from the dose-dependence studies described above. The doxycycline treatment reduced the incidence of intracranial aneurysms to 10% (P < 0.05). The incidence of intracranial aneurysms was reduced to 40% in MMP-9 knockout mice compared to the wild type mice (P < 0.05). However, there was no difference in the incidence of aneurysms between the wild type mice and MMP-2 knockout mice. KO = knockout mice.

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