Complex Hemodynamic Insult in Combination with Wall Degeneration at the Apex of an Arterial Bifurcation Contributes to Generation of Nascent Aneurysms in a Canine Model

Discussion

Further knowledge of the pathologic mechanisms underlying aneurysm generation or growth may provide the opportunity to develop mathematic models to better understand the progression of vascular diseases. Until recently, animal models have aimed to evaluate the underlying mechanisms of aneurysm formation by simulating possible aneurysm-inducing factors, such as altered hemodynamics, and have compared the morphologic or histopathologic changes with those in humans.^6⇓–8,9 In 2007, Meng et al⁷ surgically created a new bifurcation site in the canine carotid artery and reported a correlation between high WSS or WSSG and histologic insult; however, arterial wall aneurysms did not develop, and further study is required to confirm whether the observed histologic changes induced aneurysm formation. In 2008, Gao et al⁶ induced nascent cerebral aneurysms by increasing hemodynamic stress via bilateral ligation of rabbit carotid arteries. Increased hemodynamic stress triggered nascent aneurysm formation and correlated with increased blood flow in the basilar artery; however, the histologic changes could not be confirmed by traditional intra-arterial angiograms or other radiologic methods due to the tenuous blood vessel of the rabbit. Subsequent studies have reported that complex hemodynamics at bifurcation sites can induce molecular changes in a manner similar to those in human aneurysmal wall changes, including decreased expression of endothelial nitric oxide synthase, CD68 deficiency, and up-regulation of MMP-2, MMP-9, inducible NO synthase, and interleukin-1β, which is restricted to the high WSS and high WSSG areas,^5,10,11 suggesting that altered hemodynamics act at a molecular level to promote aneurysm formation.

Besides constant blood flow action, many studies have supported the suggestion that factors leading to deterioration of the arterial wall structure are the most important step in cerebral aneurysm development.¹² The apex of cerebral vasculature bifurcations differs from that in most large arteries because the median muscular layer is usually absent. In addition, cerebral vessels exhibit a fragile and/or absent external elastic lamina and are surrounded by CSF, thus rendering them susceptible to injury from mechanical forces.^3,4,12 These observations have led to the hypothesis that structural wall abnormalities and defective collagen and elastin are the major culprits in aneurysm initiation.¹³ In 2009, Nuki et al⁸ reported a novel cerebral aneurysm model by inducing hypertension and the degeneration of elastic lamina in mice and found that cerebral aneurysm generation was closely related to MMP activation⁸; however, the aneurysm site was not controllable.

In this study, we investigated the role of arterial bifurcation hemodynamic simulation combined with arterial wall degeneration and blood flow acceleration in a canine model of aneurysm generation. Specifically, we induced arterial wall degeneration via internal elastic lamina and media elastic fiber insult by using elastase to induce muscular layer degeneration after blood flow action. Using the method of Meng et al,⁷ we reconstructed bilateral CCA bifurcation apices to simulate the hemodynamic conditions and increased blood flow by unilateral CCA ligation. Because inner arterial wall elastase incubation led to a high rate of intra-arterial thrombus occurrence in preliminary experiments, we used an external wall incubation method, which selectively disrupted the internal elastic lamina and elastic fibers to simulate arterial wall degeneration. The removal of as much tunica adventitia as possible to facilitate elastase penetration was key.

Despite the fact that the blood flow pressure and velocity are similar in small animals and humans, the vessel wall structures, especially the arterial muscle layer, are very different. Consequently, aneurysms induced in small animals may not be reproducible in large animals. Here, we used a canine model because canine vessels behave more like human vessels.¹⁴ The long canine CCA (10–12 cm) provides sufficient length to construct an arterial bifurcation model. The caliber of the canine CCA and human ICA are comparable, with a constant diameter of approximately 4 mm. Moreover, the cerebral blood supply in dogs is delivered almost entirely through the vertebrobasilar system, with anastomoses between each network of the ICA,¹⁵ which helps prevent death when the bilateral CCA is temporarily occluded. Additionally, the number of cells, their composition, and the accompanying proteoglycan matrices in the canine CCA are essentially the same as in the human CCA,¹⁶ indicating that canine models may accurately predict the human biologic response to aneurysm-inducing factors.

During 24-weeks' follow-up, aneurysms were successfully induced in 5/9 elastase-treated animals in the EBG and were confirmed by both angiography and histopathology. We hypothesize that arterial wall deterioration, combined with distinct hemodynamics, effectively results in aneurysm initiation. The weakened arterial wall at the apex may adapt or respond to sustained alterations in blood flow, blood pressure, or axial stretch. Aneurysm generation or enlargement is a type of arterial wall remodelling, which may potentially involve increased inflammatory cell infiltration, increased MMP activity, extracellular matrix protein synthesis, and SMC apoptosis, to counteract the mechanical forces induced by altered blood flow.¹⁷ Inflammatory cell infiltration was detected in the media of the aneurysm walls by pan-leukocyte staining by using an anti-CD45 and MAC387 antibody, consistent with observations in animal and human intracranial aneurysms,^2,18 indicating that macrophage infiltration plays a critical role in the formation of the aneurysms observed in this model, especially during the early stages of aneurysmal formation. MMP-2 and MMP-9 were also expressed in the walls of the aneurysms, both of which possess gelatinase and collagenase activity and can degenerate the important extracellular matrix components in the walls of aneurysms, such as elastin and collagen type IV.¹⁹ Taken together, these results indicate that MMP may be released from the infiltrating macrophages and may play an important role in the progression of aneurysms.

The aneurysms had a wide neck, mainly determined by the size of the elastase-incubated area, and a small body; therefore, they can only be termed “nascent aneurysms.” However, these nascent aneurysms had histologic characteristics similar to those in human aneurysms including the following: 1) internal elastic lamina discontinuity with a reduced media elastic layer and SMC layer thickness; 2) degeneration, thinning, or interruption of the media SMC layer; and 3) significantly reduced SMC proliferation. Another feature of this model was that no thrombus formation was revealed in the aneurysm sac at angiography or during the histologic examination. This suggests that the elastase insult to the adventitia of the bifurcation apex kept the endothelial cell layer intact, to prevent thrombosis and platelet aggregation in response to expression of CD39 and secretion of nitric oxide and prostacyclin.²⁰

Blood flow is generally considered an important modulator of normal artery enlargement in response to increased flow to maintain normal shear stress.²¹ In this study, despite all models having simulated elastic fiber insults (arterial wall deterioration), nascent aneurysms were generated in only 5 of 9 models. We hypothesize that the bifurcation angle is an important factor because T-shaped bifurcations (146.8 ± 40.84°) were more likely to induce the generation of aneurysms than Y-shaped bifurcations (87.25 ± 18.87°).²² Small bifurcation angles are associated with high blood flow velocities and dispersed vortex intensities, while increased bifurcation angles result in more concentrated vortex intensities next to the apex and turbulent/disturbed laminar flow, which generates various mechanical stimuli, such as WSS and WSSG.^23,24 The quantitative analysis of the WSS and WSSG values at the bifurcation sites in our study proved that the arterial wall next to the apex was subjected to higher WSS and WSSG values before the generation of aneurysms in models with large bifurcation angles. Elevated WSS or WSSG levels disrupt the normal function of endothelial cells, stimulate gene transcription, and activate ion channels and the consequent reorganization of the cellular cytoskeleton.^5,21 The more substantial structural changes, which occur after 6 months, appear to involve the upregulation of MMP-2 and -9, both of which are implicated in aneurysmal disease.²⁵

However, this model of the carotid artery bifurcation aneurysm still does not completely represent a true cerebral aneurysm model because compared with extracranial arteries, intracranial arteries have their own specific anatomic and biologic characteristics, which could not be simulated in our model: a well-developed internal elastic lamina; a weakened tunica media and tunica adventitia, or even the absence of the tunica adventitia; reduced elastic fibers; increased proportions of SMCs; and, most important, a CSF environment.