Histology of Normal Intracranial Arteries

Intracranial arteries vary in size from fine communicating arteries (0.25 mm) to the basilar and internal carotid arteries (3 to 5 mm). Like all arteries, intracranial arteries are composed of three distinct microscopic layers: tunica intima, tunica media, and tunica adventitia, commonly referred to as the intima, media, and adventitia (Fig. 361-1A and B). The innermost layer is the intima, which is composed of a monolayer of endothelial cells on the luminal side in direct contact with blood flow, followed by a subendothelial connective tissue layer. A membrane of elastic fibers called the internal elastic lamina separates the intima from the media. The media is composed mainly of concentric sheets of smooth muscle cells and collagen fibers.^12,13 Medial collagen, apparently predominantly type III, can be stretched despite its highly aligned arrangement.¹² The outermost layer is the adventitia, which consists primarily of a complex network of type I collagen fibers, some elastin, nerves, fibroblasts, and the vasa vasorum. Adventitial collagen fibers adjacent to the media are oriented in a circumferential fashion and gradually change to a longitudinal orientation toward the outer layer.^14–16 Adventitial collagen fibrils, 50 to 100 nm in diameter,^17,18 have a wavy appearance and are unstretched at physiologic pressure levels.^16,19 In most arteries, the adventitia is separated from the media by another layer of elastic fibers called the external elastic lamina. However, this structure is not present in intracranial arteries.

FIGURE 361-1 Histology of the intracranial arteries depicting three layers of the intracranial wall (A and B) and the presence of medial gaps and pads of myointimal hyperplasia (C-E). F, Drawing showing the relative locations of medial gaps and pads of myointimal hyperplasia at a vessel bifurcation. MCA, middle cerebral artery.

(From Frosen J. The Pathobiology of Saccular Cerebral Artery Aneurysm Rupture and Repair. Helsinki: University of Helsinki; 2006:18).

A substantial portion of the connective tissue in the arterial wall is filled with extracellular matrix, a complex and dynamic meshwork of proteins and proteoglycans that provide structural support to the vessel wall, as well as participate in various biologic activities such as cell proliferation, differentiation, and migration.²⁰ Synthesized and secreted by resident cells, extracellular matrix molecules become highly organized to adapt to the functional requirements of the particular tissue. Examples of extracellular matrix components are collagen, elastin, fibronectin, laminin, nidogen/entactins, perlecan, and syndecans.

About 80% to 90% of the total arterial collagen, composed of collagen types I and III, consists of fibrillar collagen, which provides tensile strength to blood vessels. The defining structural feature of collagen is a triple-stranded helical structure (designated procollagen) of three polypeptide chains, referred to as α chains. Once secreted, propolypeptides of procollagen are removed by proteolytic enzymes. This allows the formation of higher order polymers called collagen fibrils (10 to 30 nm in diameter), which often aggregate into even larger bundles called collagen fibers (500 to 3000 nm in diameter). The remaining 10% to 20% of arterial collagen (types IV, V, VI, and VIII)²¹ is composed of network-forming collagen that gives rise to net-like structures, such as in the basement membrane.

Elastin is the core component of elastic fibers and it provides resilience to blood vessels. On secretion, elastin precursor molecules become highly cross-linked to one another and form networks of elastin fibers and sheets. Microfibrils, composed of glycoproteins, are thought to provide scaffolding for elastin molecules and are subsequently displaced as elastin is deposited into the growing fiber. Elastin makes up about 90% of elastic fibers, with microfibrillar glycoproteins accounting for the remaining 10%.²²

Fibronectin is a multifunctional glycoprotein that provides stability to vessel walls and is involved in intercellular contact and repair mechanisms.^23,24 Fibronectins connect cells with collagen fibers, thereby allowing cells to move through the extracellular matrix. They bind collagen and cell surface receptors called integrins, which causes reorganization of the cell cytoskeleton and cell movement. Fibronectins are secreted in an inactive form. Binding to integrins allows them to dimerize and become active. Fibronectins also help at the site of tissue injury by binding to platelets during blood clotting and facilitating movement of cells to the affected area during wound healing.

Laminins, a family of heterotrimeric glycoproteins that are usually found in basement membranes, function in migration, adhesion, and cell signaling.^25,26 They are formed from the assembly of three subunits (α, β, and γ). Several laminin isoforms are derived from the combination of five α, four β, and six γ subunits. Vascular endothelial cells have been shown to produce the laminin-8 and laminin-10 isoforms.^25,27,28

In 1930, Forbus described the presence of discontinuities in the smooth muscle layer of arterial vessels, especially at the apex of vessel bifurcations. For many years, these medial gaps were believed to represent congenital medial defects that contribute to the development of intracranial aneurysms.²⁹ In 1983, Stehbens demonstrated that the so-called medial defects could be visualized under light microscopy and that they are actually physiologic intersections of two muscle layers rather than defects.³⁰ This finding is in agreement with more recent studies showing that highly organized tendon-like collagen is consistently found in the region of the medial gap, which would provide strength and stability to the vessel, and is inconsistent with the concept of defect.¹⁷ Accordingly, experimentally induced intracranial aneurysms in rats were shown not to originate at the site of these medial gaps.³¹ The histologic characteristics of these medial gaps are shown in Figure 361-1.³²

Histology of Intracranial Arteries from Patients with Aneurysms

In contrast to the highly ordered structure of a normal arterial wall, aneurysms have a disorganized wall structure with less distinct layers. A common feature of intracranial aneurysm walls is loss or rupture of the internal elastic lamina (Fig. 361-2A).^33–36 Other characteristics include intimal thickening resembling myointimal hyperplasia, muscle fiber disarray, depletion of cellular components (Fig. 361-2B and C), and irregularities in the luminal surface (Fig. 361-3).^{33,34,36–42} Atherosclerotic lesions have also been identified in some aneurysms.^38,43–45 Immunohistochemical studies on 18 intracranial aneurysm specimens showed intermingling of collagen type I and fibronectin throughout the aneurysm wall. This finding is in contrast to parent and normal arteries, in which collagen and fibronectin are limited to the adventitia and media, respectively.⁴⁶ Expression of laminin, collagen types III and IV, and the angiogenic growth factor transforming growth factor-α has been found to be decreased in some aneurysm samples when compared with normal vessels (Fig. 361-4).⁴⁷

FIGURE 361-2 Histologic sections of human paraffin-embedded intracranial aneurysm specimens stained with orcein (A) or hematoxylin-eosin (B and C). Abrupt termination of the internal elastic lamina (long arrow) and the tunica media (short arrow) is seen at the neck of an aneurysm (A). Absence of inflammation and loss of cellular elements are noted at 40× magnification (B) and are more evident at higher magnification (C). Arrows in (B) indicate the luminal surface of the aneurysm wall.

(From Abruzzo T, Shengelaia GG, Dawson RC 3rd, et al. Histologic and morphologic comparison of experimental aneurysms with human intracranial aneurysms. AJNR Am J Neuroradiol. 1998;19:1309).

FIGURE 361-3 Scanning electron microscopy of the intimal surface of ruptured human intracranial aneurysms (A and C) and a control middle cerebral artery (B). The surface of the aneurysm specimens is more rugged and uneven than the surface of the control sample. Original magnification, ×3400 in A and B, ×6000 in C.

(Reprinted from Hassler O. Scanning electron microscopy of saccular intracranial aneurysms. Am J Pathol. 1972;68:511, with permission from the American Society for Investigative Pathology.)

FIGURE 361-4 Immunostaining for structural proteins in control arteries and in unruptured and ruptured human intracranial aneurysms.

(From Kilic T, Sohrabifar M, Kurtkaya O, et al. Expression of structural proteins and angiogenic factors in normal arterial and unruptured and ruptured aneurysm walls. Neurosurgery. 2005;57:997.)

Changes in the medial smooth muscle cell phenotype have also been reported in intracranial aneurysms. In control cerebral arteries, α-smooth muscle actin, desmin, and the smooth muscle myosin heavy-chain (MHC) isoforms SM1 and SM2 were strongly expressed, whereas another MHC isoform, SMemb, was weakly expressed. In aneurysm walls, no expression of desmin was observed. In some aneurysms, SMemb expression was higher or SM2 expression was lower than in control arteries. Desmin is the major intermediate filament of skeletal, cardiac, and smooth muscle tissue⁴⁸ and is believed to be important for strengthening and maintaining the integrity of smooth muscle cells.⁴⁹ These observations suggest that a phenotypic switch in smooth muscle cells is associated with vascular remodeling in the aneurysm wall.⁵⁰

Histologic differences have been noted between ruptured and unruptured aneurysms. A denser expression of fibronectin has been observed in ruptured aneurysms than in unruptured aneurysms.⁴⁷ Ruptured walls appear to have more pronounced endothelial damage and loss of wall density.^41,51–53 In a study involving 44 ruptured and 27 unruptured aneurysms, Kataoka and colleagues found that unruptured aneurysms had thick walls resembling myointimal hyperplasia whereas ruptured aneurysms had thin, hyalinized, and hypocellular walls.⁴¹ In a series of 66 intracranial aneurysm samples, Frosen and associates identified four histologic aneurysm wall types and described morphologic changes in the aneurysm walls that correlated with rupture. These wall types were endothelialized wall with linearly organized smooth muscle cells (42% ruptured) (Fig. 361-5A), thickened wall with disorganized smooth muscle cells (55% ruptured) (Fig. 361-5B), hypocellular wall with myointimal hyperplasia (64% ruptured) (Fig. 361-5C), and thin, hyalinized, and hypocellular wall (100% ruptured) (Fig. 361-5D).⁵¹

FIGURE 361-5 Four aneurysm wall types—type A, type B, type C, and type D—associated with rupture (A-D, respectively). Antibodies used for immunostaining are indicated in each panel. HE, hematoxylin-eosin; MHC, myosin heavy chain.

(From Frosen J, Piippo A, Paetau A, et al. Remodeling of saccular cerebral artery aneurysm wall is associated with rupture: histological analysis of 24 unruptured and 42 ruptured cases. Stroke. 2004;35:2287.)

Structural and biochemical changes have also been noted in the nonaneurysmal tissue of aneurysm patients. In a study of the intracranial arteries of 70 normal individuals and 35 patients with intracranial aneurysms who died of aneurysm rupture, a dense, uniformly distributed network of reticular fibers surrounding the smooth muscle cells was observed in the media of normal individuals. This pattern did not appear to change with age, at least until the age of 50. In contrast, a decreased number of reticular fibers was observed in the arteries of aneurysm patients, all of whom died before the age of 50.⁵⁴ Similar results were obtained in two other independent investigations.^55,56 Aside from decreased numbers, irregular distribution and shorter reticular fiber length were also noted.^55,56 Biochemical studies have shown that the skin fibroblasts and intracranial and temporal arteries of some aneurysm patients are deficient in collagen type III relative to collagen type I.^57–60 These observations led to the hypothesis that “intrinsic” abnormalities in the walls of intracranial arteries lead to conditions that favor the formation and rupture of intracranial arteries.

Proteolysis and Intracranial Aneurysms

Extracellular matrix components are constantly being synthesized and degraded. When compared with normal intracranial arteries, aneurysm tissue, especially ruptured aneurysm tissue, displays increased activity or expression (or both) of matrix-degrading proteases that regulate remodeling of the arterial wall. Prominent in aneurysm walls are matrix metalloproteinase-2 (MMP-2) and MMP-9, which are classified as gelatinases and are capable of degrading both elastin and denatured collagen. In a series of 23 patients with intracranial aneurysms, Bruno and associates found focal areas of gelatinase activity attributable to MMP-2 and MMP-9 in both ruptured and unruptured aneurysms. Increased protease activity correlated with increased expression of MMP-2, plasmin (Fig. 361-6), and membrane-type MMP,¹¹ with the latter two being involved in activation of MMP-2. Increased serum levels of elastase have also been observed in aneurysm patients.^64,65