suggest that collagen type VI has a role similar to that of type V in maintaining the structural integrity of the collagenous core. The fact that there were no differences in intensity or pattern of the collagen stainings between normal and necrotizing scleritis suggests that proteoglycans are the Þrst extracellular matrix components to be degraded in necrotizing scleritis. Proteoglycan degradation may enhance intracellular and extracellular mechanisms of collagen Þbril depletion.

3.Glycoproteins. Fibronectin and vitronectin showed subtle, granular, patchy positivity in normal and inßamed sclera. Laminin was absent in extravascular sclera, normal or inßamed, but was dramatically represented in vessel walls.

Vessels

The area of active scleral inßammation shows old and new vessels, many of which display neutrophil and lymphocyte inÞltration within and around the vessels, with occasional thrombosis [28, 30, 39, 40].

Our own studies on scleral vasculature in 26 scleral specimens from patients with noninfectious necrotizing scleritis were accomplished by use of histopathological and direct immunoßuorescence techniques. Specimens examined by the direct immunoßuorescence technique were incubated with ßuoresceinand rhodamineconjugated goat antibodies directed against human immunoglobulins IgG, IgA, IgM, IgD, and IgE, complement components C3 and C4, and albumin [28] (as a negative control for vascular positivity) (Organon Teknika-Cappel ScientiÞc, West Chester, PA) (Table 5.6). Goat anti-human collagen IV (Biodesign International, Kennebunkport, ME) was used as a positive control for vessels. Fluorescence microscopy was performed on a Zeiss (Thornwood, NY) Photomic III ßuorescence microscope. All scleral specimens but one (96%) showed an inßammatory microangiopathy characterized by neutrophilic inÞltration in and around the vessel wall, as visualized by histopathological technique (Fig. 5.12), or immunoreactant deposition on the vessel wall, as visualized by the immunoßuorescence technique (Fig. 5.13). Inßammatory microangiopathy as

Table 5.6 Antibody panel used in immunoßuorescence studies of scleral specimens

aIndirect immunoßuorescence technique

Fig. 5.12 Inßammatory microangiopathy. Note the neutrophil inÞltration in and around the wall of the conjunctival vessel

Fig. 5.13 Inßammatory microangiopathy, as visualized by immunoßuorescence microscopy. The antibody used in this specimen is anti-IgG antibody, and the photomicrograph shows the presence of IgG in the vessel walls, with the bright ßuorescence in the area of the basement membrane of the vessel wall (MagniÞcation, ×64)

detected by histopathological technique was found in 74% of the scleral specimens; in addition to the neutrophilic inÞltration in and around the vessel wall, vessel occlusion was often found within necrotic areas. Inßammatory microangiopathy detected by the immunoßuorescence technique was found in 94% of the scleral specimens. These data show that inßammatory microangiopathy is highly associated with the most severe and destructive type of scleritis, necrotizing scleritis; the immunoßuorescence technique increases the sensitivity of detection of inßammatory vascular damage in necrotizing scleritis.

Episcleral vessels and perforating intrascleral vessels, as capillaries and postcapillary venules, have a wall that consists of endothelial cells attached to an underlying basement membrane secreted by them, and a discontinuous layer of pericytes. Our studies on immunolocalization of connective tissue components in normal scleral blood vessels showed the presence of collagen types IV, V, and VI, heparan sulfate and chondroitin sulfate, Þbronectin, and laminin in endothelial cell basement membranes. Necrotizing scleritis specimens showed marked proliferation of scleral blood vessels with positive stainings for the same connective tissue components as found in normal scleral blood vessels (Fig. 5.14a, b).

Eight of 13 (61%) scleral specimens from patients with noninfectious recurrent diffuse or nodular scleritis showed inßammatory microangiopathy by histopathological or immunoßuorescent techniques. Neutrophilic inÞltration in and around the vessel wall was detected in 28% of the scleral specimens; vessel occlusion was not detected in any. Immunoreactant deposition on vessel walls was detected in 80% of the scleral specimens. These data show that inßammatory microangiopathy is less associated with nonnecrotizing scleritis than with necrotizing scleritis and that the immunoßuorescence technique increases the sensitivity of detection of inßammatory vascular damage in nonnecrotizing scleritis. Vasculitis/inßammatory microangiopathy may also be detected in the anterior ciliary arteries in enucleation specimens from patients with scleritis (Fig. 5.15).

Fig. 5.14 (a) Necrotizing scleritis, as visualized by immunoßuorescence microscopy (MagniÞcation, ×40). The antibody used is anti-collagen type IV. Anti-collagen type IV antibodies stain vascular basement membranes, and this specimen shows, compared with (b), a large number of blood vessels. (b) Normal sclera (same technique and magniÞcation as in panel a). Note the paucity of vessels in this specimen, particularly when compared to that of the patient with necrotizing scleritis (a)

Fig. 5.15 Scleral biopsy of a patient with scleritis. Note the inßammatory microangiopathy, with clustering of inßammatory cells around the vessel. Because the vessel lacks a true vascular wall, however, the criteria typically used by general pathologists to declare the presence of a true vasculitis cannot be used in analyzing these specimens (MagniÞcation, ×60; hemaloxylineosin stain)