Critical role of TNF-α in cerebral aneurysm formation and progression to rupture

Abstract

Background: Alterations in TNF-α expression have been associated with cerebral aneurysms, but a direct role in formation, progression, and rupture has not been established.

Methods: Cerebral aneurysms were induced through hypertension and a single stereotactic injection of elastase into the basal cistern in mice. To test the role of TNF-α in aneurysm formation, aneurysms were induced in TNF-α knockout mice and mice pretreated with the synthesized TNF-α inhibitor 3,6'dithiothalidomide (DTH). To assess the role of TNF-α in aneurysm progression and rupture, DTH was started 6 days after aneurysm induction. TNF-α expression was assessed through real-time PCR and immunofluorescence staining.

Results: TNF-α knockout mice and those pre-treated with DTH had significantly decreased incidence of aneurysm formation and rupture as compared to sham mice. As compared with sham mice, TNF-α protein and mRNA expression was not significantly different in TNF-α knockout mice or those pre-treated with DTH, but was elevated in unruptured and furthermore in ruptured aneurysms. Subarachnoid hemorrhage (SAH) occurred between 7 and 21 days following aneurysm induction. To ensure aneurysm formation preceded rupture, additional mice underwent induction and sacrifice after 7 days. Seventy-five percent had aneurysm formation without evidence of SAH. Initiation of DTH treatment 6 days after aneurysm induction did not alter the incidence of aneurysm formation, but resulted in aneurysmal stabilization and a significant decrease in rupture.

Conclusions: These data suggest a critical role of TNF-α in the formation and rupture of aneurysms in a model of cerebral aneurysm formation. Inhibitors of TNF-α could be beneficial in preventing aneurysmal progression and rupture.

Figure 1

Blood pressure analysis. Blood pressure was elevated 1 week after aneurysm induction, but was not significantly different between mice pre-treated with DTH, TNF-α knockout mice, and mice receiving vehicle alone. DTH, 3,6'dithiothalidomide.

Figure 2

The role of TNF-α in the formation of intracranial aneurysms. (A) Cerebral aneurysm formation was significantly increased in mice receiving vehicle as compared to TNF-α knockout mice and those pre-treated with DTH. (B) The incidence of aneurysm rupture was also significantly increased in mice receiving vehicle versus TNF-α knockout mice and those pre-treated with DTH. (C) Kaplan-Meier analysis demonstrates that aneurysm rupture occurred between 7 and 21 days following aneurysm induction, and mice receiving vehicle were more likely to have ruptured aneurysms as compared to TNF-α knockout mice and those pre-treated with DTH. DTH, 3,6'dithiothalidomide.

Figure 3

Cerebral aneurysm formation and rupture and TNF-α expression. Representative gross images of normal cerebral blood vessels in (A) sham operated mice, (B) unruptured aneurysms, and (C) ruptured aneurysms. H & E staining of normal cerebral blood vessels in sham operated mice demonstrates two to three layers of smooth muscle cells and a single, thin, continuous layer of endothelial cells (A2 to 3). In both unruptured cerebral aneurysms (B2 to 3) and ruptured aneurysms (C2 to 3) there are thin and thick areas of vascular wall. In unruptured aneurysms in the areas of thin vascular wall, there is intact endothelial and smooth muscle cell layers, but this is discontinuous in the areas of thick vascular wall. In ruptured aneurysms these areas are sometimes discontinuous in both thick and thin areas of the vascular walls. Trichrome staining of normal cerebral blood vessels in sham operated mice (A4 to 5) demonstrates one layer of elastic lamina. This is disorganized in unruptured aneurysms (B4 to 5) and ruptured aneurysms (C2 to 3). TNF-α (green) expression is nearly non-existent in sham operated mice (A6 to 7). The expression of TNF-α (green) is decreased in sham mice (A6 to 7) as compared to unruptured (B6 to 7) and furthermore ruptured aneurysms (C6 to 7). SMC-22α (red) is observed in sham operated mice (A6) and co-localizes with TNF-α (green) to appear yellow in unruptured (B6) and ruptured aneurysms (B7). Normal sized cerebral blood vessels also stain only red for SM-22α in vascular segments adjacent to cerebral aneurysms (B6). Macrophages are nearly non-existent in sham operated mice (A7). Macrophages (red) co-localize with TNF-α (green) and appear yellow in unruptured aneurysms (B7) and more so in ruptured aneurysms (C7). Nuclei are stained blue with DAPI. DAPI, 4',6-diamidino-2-phenylindole; H & E, hematoxylin and eosin; SM-22α, smooth muscle cell 22 alpha.

Figure 4

TNF-α mRNA expression. As compared with sham mice receiving vehicle, real-time PCR demonstrated no significant difference in TNF-α mRNA expression of cerebral blood vessels from TNF-α knockout mice or those pre-treated with DTH, but TNF-α was significantly elevated in unruptured and furthermore in ruptured aneurysms. DTH, 3,6'dithiothalidomide.

Figure 5

Blood pressure analysis in delayed DTH treatment. Blood pressure was elevated 1 week after aneurysm induction, but was not significantly different between mice treated with DTH starting on day 7 and mice receiving vehicle alone. DTH, 3,6'dithiothalidomide.

Figure 6

Aneurysm stabilization with DTH. (A) There was no significant difference in the overall incidence of aneurysm formation between cohorts treated with DTH and those treated with vehicle. (B) The incidence of rupture was significantly decreased in those treated with DTH as compared to vehicle. (C) The rate of cerebral aneurysm rupture in those with cerebral aneurysm formation was significantly decreased in those treated with DTH as compared with vehicle. (D) After eliminating those animals without aneurysm formation, Kaplan-Meier analysis demonstrated that those receiving delayed DTH treatment were 5.8 times (95% CI 1.3 to 25.5, P = 0.020) less likely to have aneurysmal rupture than those receiving vehicle. DTH, 3,6'dithiothalidomide.