Pharmacological stabilization of intracranial aneurysms in mice: a feasibility study

Abstract

Background and purpose: An increasing number of unruptured intracranial aneurysms are being detected, partly due to the increased use of brain imaging techniques. Pharmacological stabilization of aneurysms for the prevention of aneurysmal rupture could potentially be an attractive alternative approach to clipping or coiling in patients with unruptured intracranial aneurysms. We have developed a mouse model of intracranial aneurysm that recapitulates key features of intracranial aneurysms. In this model, subarachnoid hemorrhage from aneurysmal rupture causes neurological symptoms that can be easily detected by a simple neurological examination. Using this model, we tested whether anti-inflammatory agents such as tetracycline derivatives, or a selective inhibitor of matrix metalloproteinases-2 and -9 (SB-3CT), can prevent the rupture of intracranial aneurysms.

Methods: Aneurysms were induced by a combination of induced hypertension and a single injection of elastase into the cerebrospinal fluid in mice. Treatment with minocycline, doxycycline, or SB-3CT was started 6 days after aneurysm induction. Aneurysmal rupture was detected by neurological symptoms and confirmed by the presence of intracranial aneurysms with subarachnoid hemorrhage.

Results: Minocycline and doxycycline significantly reduced rupture rates (vehicle versus doxycycline=80% versus 35%, P<0.05; vehicle versus minocycline=73% versus 24%, P<0.05) without affecting the overall incidence of aneurysms. However, SB-3CT did not affect the rupture rate (62% versus 55%, P=0.53).

Conclusions: Our data established the feasibility of using a mouse model of intracranial aneurysm to test pharmacological stabilization of aneurysms. Tetracycline derivatives could be potentially effective in preventing aneurysmal rupture.

Figure 1

A: Incidence of unruptured and ruptured aneurysms at 7 and 28 days after aneurysm induction. At 7 days after aneurysm induction, 63% of the mice had unruptured aneurysms, but none of the mice had ruptured aneurysms. Over 28 days period, 40% of the mice developed subarachnoid hemorrhage from ruptured aneurysms. At 28 days after aneurysm induction, 40% of the mice had unruptured aneurysms. The overall incidence of aneurysms was similar between mice that were sacrificed at day 7 and mice that that were followed for 28 days after aneurysm induction, indicating that most aneurysm formations precedes aneurysmal rupture. B: Cumulative symptom free curve. The cumulative symptom free curve was plotted for the mice that had aneurysm to show a time course of the onset of symptoms. Mice that did not reveal any aneurysm formation were excluded from this graph. Aneurysmal rupture started occurring 7 days after aneurysm induction. The overall rupture rate was 50%. C: Representative ruptured and unruptured aneurysms. D: Experimental protocol to test pharmacological treatments for the prevention of aneurysmal rupture.

Figure 2. In-situ zymography of ruptured and unruptured intracranial aneurysms

A: Control cerebral artery showed scant matrix metalloproteinase (MMP) activity. B: An unruptured aneurysm showed MMP activity. However, the adjacent parent artery (middle cerebral artery) showed little MMP activity. C: A ruptured aneurysm revealed abundant MMP activity. There was a general trend that MMP activity was more prominent in ruptured aneurysms.

Figure 3. Effects of doxycycline on aneurysmal rupture

A: Incidence of aneurysms. Although there was no difference in the overall incidence of aneurysms (both ruptured and unruptured) between the vehicle and doxycycline group (68 vs. 68%, P = 0.62), the incidence of ruptured aneurysms was significantly lower in the doxycycline group than in the vehicle group (24 vs. 55%, P < 0.05). B: Rupture rate. The doxycycline treatment significantly reduced the rupture rate compared to the vehicle treatment (80 vs. 35%, P < 0.05). C: Symptom-free curve (Kaplan-Meier analysis curve). A log-rank test revealed a significant reduction of aneurysmal rupture by the doxycycline treatment (P < 0.05). Mice that did not develop aneurysms were excluded from the survival analyses.

Figure 4. Effects of minocycline on aneurysmal rupture

A: Incidence of aneurysms. There was no difference in the overall incidence of aneurysms (both ruptured and unruptured) between the vehicle and minocycline group (71 vs. 65%, P = 0.45). However, the incidence of ruptured aneurysms was significantly lower in the minocycline group than in the vehicle group (16 vs. 52%, P < 0.05). B: Rupture rate. Minocycline treatment significantly reduced the rupture rate compared to the vehicle treatment (73 vs. 24%, P < 0.05). C: Symptom-free curve. Log-rank test for those mice that had aneurysms revealed a significant reduction of aneurysmal rupture by the minocycline treatment (P < 0.05).

Figure 5. Effects of SB-3CT on aneurysmal rupture

A: Incidence of aneurysms. There was no difference in the overall incidence of aneurysms (both ruptured and unruptured) or the incidence of aneurysmal rupture between the vehicle and SB-3CT group. B: Rupture rate. The treatment with SB-3CT, a selective inhibitor for MMP-9 and MMP-2, did not affect the rupture rate. C: Symptom-free curve. Log-rank test for those mice that had aneurysms revealed no significant difference between the vehicle group and SB-3CT group.