Gregory S. Hageman

Localization of carbonic anhydrase IV in a specific capillary bed of the human eye

Carbonic anhydrase (CA) activity plays an important role in controlling aqueous humor production in the eye and in regulating intraocular pressure. Prior studies identified the soluble isozymes CA II and CA I in the human eye and also suggested a distinct membrane-associated CA. We used an antibody to CA IV, the membrane-anchored isozyme from human lung, to study CA IV in eye tissues and to compare its distribution with that of CA II. We found intense immunostaining for CA IV associated with endothelial cells of one specific uveal capillary bed, the choriocapillaris. CA IV was not detected in endothelial cells of the contiguous capillaries of the iris or in endothelial cells of other vessels. Immunoreactivity for CA IV was also intense in epithelial and fiber cells of the lens but was not detectable in the neuroretina, the ciliary process (except for capillaries), and the cornea, all sites where immunostaining with anti-CA II antibody was intense. These studies indicate that the membrane-associated CA in human eye, which was suspected from histochemical studies, is CA IV. Defining the physiological role of this ocular isozyme remains a challenge.

Characterization of Drusen-associated Glycoconjugates

BACKGROUND Drusen are extracellular deposits that accumulate between the basal lamina of the retinal pigment epithelium and the elastic lamina of Bruch membrane in aging human eyes. Although specific types of drusen are recognized as significant risk factors for the development of both the atrophic and exudative forms of age-related macular degeneration, few studies have focused on defining their molecular composition. As an initial step toward identifying the molecular composition of drusen, assessing the biochemical relation between hard and soft drusen, and identifying potential target molecules for detecting drusen clinically, the authors have analyzed their carbohydrate composition using lectin histochemistry. METHODS Sections of eyes from human donors containing a spectrum of hard and soft drusen were screened with a battery of 22 fluorescein-conjugated lectins. RESULTS A specific subset of six lectins bind drusen intensely. No significant differences in lectin binding are observed between any subclass of hard and soft drusen. Some drusen exhibit homogeneous, uniform labeling, whereas others show asymmetrical, heterogeneous distribution of glycoconjugates. CONCLUSION This study shows that glycoconjugates containing specific carbohydrate residues are present in all classes of hard and soft drusen examined. The observation that hard and soft drusen are bound by the same lectins suggests that they may be related compositionally. Identification of the drusen-associated glycoconjugates shown in this investigation will facilitate studies of drusen genesis and their involvement in the pathogenesis of age-related maculopathy. They may also provide a basis for developing avenues of therapeutic intervention.

Histological and radiological analyses of hydroxyapatite orbital implants in rabbits.

Summary To date, only anectodal clinical data exist pertaining to the histological changes of hydroxyapatite within an enucleated socket. This study was conducted to determine the histological and radiological changes in a coralline hydroxyapatite sphere placed into the central socket, in a controlled fashion. Rabbits underwent simple enucleation with implantation of an autologous sclera-wrapped hydroxyapatite spheres with extraocular muscle reattachment. Preoperatively, the mineral density of each sphere was determined using quantitative computed tomography (CT) that was repeated 2− and 6 weeks postoperatively. The implants were harvested at 2− and 6 weeks and submitted for light and electron microscopic analysis. The results demonstrated a uniform influx of fibrovascular tissue that did not reach the center of the implant, even at 6 weeks. A marked mixed-cell inflammatory response was noted at the interface between the fibrovascular tissue and the hydroxyapatite. Giant cells were noted only at the scierai windows. This study demonstrated that the early response to hydroxyapatite implants was fibrovascular ingrowth with mixed-cell inflammation. These histological observations correlated with findings observed with quantitative CT. Quantitative CT appears to be an ideal modality for observing the early temporal tissue density changes in hydroxyapatite implants.

CHARACTERIZATION OF A CENTRAL CORNEAL CLOUDINESS SHARING FEATURES OF POSTERIOR CROCODILE SHAGREEN AND CENTRAL CLOUDY DYSTROPHY OF FRANCOIS

A 56-year-old black woman with full-thickness mosaic pattern central corneal cloudiness, similar in appearance to central cloudy dystrophy and posterior crocodile shagreen, underwent corneal transplantation. Atypical features included decreased vision, photophobia, and epithelial involvement, with occasional foreign body sensation. Numerous 0.5-2.0-µm-diameter lacunae were present in the corneal stroma and Bowmans layer, and a saw-toothed lamellar pattern was often evident in the corneal stroma. Soybean agglutinin (SBA), a lectin that binds N-acetyl-galactosamine residues, bound diffusely to stromal foci exhibiting similar size and distribution to the lacunae observed by electron microscopy. An absence of histochemically detectable lipid associated with these lacunae suggests that SBA reacted with glycoconjugates other than glycolipids. Biochemical analyses revealed similar contents of keratan sulfate, chondroitin/dermatan sulfate, and collagen as in normal controls, suggesting that the SBA binding moieties are associated with a glycoprotein or proteoglycan that is structurally or compositionally different from those found in normal cornea. This patient may represent an extreme variant of Vogts or Francosis central corneal clouding or a previously undescribed corneal dystrophy.

Histochemical Comparison of Ocular “Drusen” in Monkey and Human

Age-related macular degeneration (AMD), the leading cause of irreversible blindness in the industrialized world, is characterized, in part, by the presence of drusen and basal laminar deposits (BLD) in the macula. Drusen are extracellular deposits which form between the retinal pigment epithelial (RPE) basal lamina and the inner collagenous zone of Bruch’s membrane. Although the relationship between macular drusen and the development of AMD has been established in a number of studies (1–4), relatively little is known about their composition or origin. Thorough characterization of these age-related deposits has been hampered by a paucity of suitable human donor tissues and the lack of an appropriate animal model of the disease.

Changes in Müller cell plasma membrane specializations during subretinal scar formation in the rabbit

The aim of this study was to identify changes in Müller cell plasma membrane specializations during experimentally induced subretinal gliosis in rabbits. When rabbits are dosed with sodium iodate, large expanses of retinal pigment epithelium and photoreceptors are destroyed. They are replaced by a subretinal scar consisting mainly of the ascending processes of Müller cells. These processes transform from the slender, highly polarized structures seen in normal animals into irregular processes that form a glia limitans along the basement membrane of the pigment epithelium, left bare following its degeneration. As the scar processes extend through the subretinal space and contract this basement membrane, they undergo dramatic changes in shape that are especially apparent in three-dimensional computer reconstructions of serial thick sections examined by high-voltage electron microscopy. Other changes involve the intercellular junctions and apical microvilli normally associated with the external limiting membrane. These structures become scattered over the surfaces of the ascending processes and are eventually lost. Loss of microvilli is associated with disappearance of immunostaining for a specific glycoconjugate normally associated with the microvillar plasma membrane. The observations document profound changes in Müller cell structural and functional polarity during subretinal scar formation.

Chondroitinase as a Vitreous Interfactant: Vitreous Disinsertion in the Human

Despite the initial discovery and testing of chondroitinase as a pharmacologic vitreolysis agent that began nearly three decades ago, contractual restrictions in the initial years and intellectual drift in more recent years have limited the peer-reviewed publication of most of the animal (monkey) and human studies that evaluated this promising agent. Detailed investigator, institutional, corporate, and registered FDA regulatory documents that are available to us provided the basis for the majority of this chapter. Two devastating floods of Dr. Gregory Hageman’s data room at the University of Iowa, however, reduced the number and variety of images available from which to provide supplementary visual illustration of the human results that will be discussed.