Endogenous hyaluronan in corneal disease.

PURPOSE: Hyaluronan (HA) is a disaccharide polymer capable of binding considerable amounts of water. It is present in trace amounts on the cornea endothelium, and it is not normally found in the epithelium or stroma. A specific histochemical stain was used to test for HA in a wide variety of corneal disorders. METHODS. Eighty-six human corneal tissue specimens were examined histochemically for HA. The material consisted of 84 full-thickness corneal buttons, one epithelium scraping, and one pterygium. Cases were analyzed according to the patient's sex, age, diagnosis, and localization of HA staining. RESULTS: The corneal tissue specimens came from 47 women and 39 men, average age 59 years. Fifty-seven percent of the specimens displayed abnormal HA. HA was visualized in Fuch's dystrophy, keratoconus, infections, regrafts, mechanical and chemical trauma, post-excimer ablations, dystrophies, degenerations, pseudophakic bullous keratopathy, congenital opacities, Stevens-Johnson syndrome, and others. Staining was variously seen in the epithelium, stroma, and endothelium, with intensity of staining ranging from trace amounts to extremely heavy. CONCLUSIONS: Endogenous hyaluronan production is seen in virtually the entire spectrum of corneal disorders. The presence of HA was most often associated with dividing, migrating, or fibroblast-like cells and probably represents a nonspecific tissue response to wounding. Its production is biochemically distinct from that of normally present proteoglycans. The abnormal presence of HA may reduce corneal transparency by disrupting the normal spacing between collagen fibrils, creating focal changes in the index of refraction, and altering the normal flow of solutes through the cornea.

Hyaluronic acid in the rabbit cornea after excimer laser superficial keratectomy.

Hyaluronic acid (HA) is not normally found in the corneal stroma. Rabbit corneas were examined for the presence of stromal HA after excimer laser treatment. One eye in each of 28 rabbits received a 60 microns deep superficial keratectomy with the excimer laser. After 1, 8, 21, and 60 days, the corneas were analyzed by quantitative and histochemical methods specific for HA. A statistically significant increase in the HA concentration compared to the baseline amount in the untreated fellow eye was seen at 8, 21, and 60 days. HA was visualized histochemically in the anterior stroma of the excimer-treated eyes at all times tested. The presence of HA after excimer surgery may influence the hydration, thickness, and transparency of the cornea. The reactive production of HA in the stroma may represent a nonspecific corneal tissue response to injury.

Evidence of hyaluronic acid and hyaluronic acid binding sites on human corneal endothelium.

A highly specific hyaluronic acid (HA) recognizing protein (HABR) was used to study whether the human corneal endothelium is covered by HA and to quantify the amount. Tritiated high molecular weight HA was used to determine the capacity of the human endothelium to bind exogenous HA. Human corneas were obtained from keratoconus patients having corneal transplantation and from postmortem eyes. The corneas were immersed in a 4% formaldehyde solution containing 1% cetylpyridine chloride for histochemistry, frozen for biochemistry, or used for 3H-HA (Mr 3 x 10(6) binding. For the biochemical determinations, 125I-labeled HABR was used. Tritiated HA was used for the binding experiment. A specific layer of HA covering the endothelial cells of the corneal buttons was demonstrated. The biochemical analysis also revealed the presence of HA. Finally, the human endothelial cells had specific hyaluronic acid binding sites.

Reactive formation of hyaluronic acid after small and large lens injury.

The reactive production of hyaluronic acid was studied in the rabbit lens following a 4 mm in diameter anterior lens wound, and following a large lens wound (extracapsular cataract extraction). Furthermore, human post-mortem after-cataract specimen were also studied. The hyaluronic acid was localized histochemically using a new specific technique. Hyaluronic acid in the small wound was localized in the primary sealing plug that precedes the reepithelialization and new capsule formation. In the large wound hyaluronic acid was found around the cells proliferating on the posterior capsule. The same was true in the human after-cataract specimen. It was concluded that the primary wound sealing and the posterior capsule cellular outgrowth constitute similar cellular responses.

Reactive formation of hyaluronic acid in the rabbit corneal alkali burn.

The presence and distribution of reactively formed hyaluronic acid (HA) was assessed in the rabbit cornea following a penetrating alkali burn. The injury was inflicted by applying a round, 5.5 mm, filter paper soaked in 1 N NaOH centrally on the cornea for 60 seconds. Biochemical analysis revealed a significant increase of HA two weeks after injury, a peak concentration after 1 month, and a decrease again at three months. Histochemical analysis revealed the presence of hyaluronic acid in the healing epithelium, in the repopulating keratocytes/fibroblasts, and in the cells forming the retrocorneal membrane. Extensive amounts filled lacunae in the stroma as well as the spaces between collagen lamellae. A slow restoration of normal appearing corneal stroma took place at the periphery. Significant staining for HA in lacunae was present centrally in the wound after three months.

Visualization of hyaluronic acid in the anterior segment of rabbit and monkey eyes.

Hyaluronic acid was visualized using a new histochemical technique: the hyaluronic acid-binding region of the cartilage proteoglycan was isolated, linked with biotin and used in histological sections as a ligand for hyaluronic acid. Staining was performed using the avidin-biotin-peroxidase technique. The presence of hyaluronic acid in the anterior segments of rabbit and monkey eyes was studied in fresh-frozen, as well as in fixed paraffin sections; addition of cetyl-pyridinium chloride to the fixative was essential. In both species intense staining was seen in the stroma of the conjunctiva, the connective tissue of the ciliary processes, and on the apical membranes of the corneal endothelium. Species differences were observed in both conventional and unconventional outflow pathways. In rabbits, the tissue located directly adjacent to the aqueous plexus and the connective tissue surrounding the poorly developed ciliary muscle were stained. In monkeys, only the anterior, non-filtering part of the trabecular meshwork and the outer wall of Schlemm's canal showed intense staining in paraffin sections. In frozen sections, the inner wall (cribriform region of the trabecular meshwork) was also stained. The spaces between the anterior part of the ciliary muscle bundles remained almost unstained.

Hyaluronate binding to intact corneas and cultured endothelial cells.

Sodium hyaluronate (HA) protects the corneal endothelium during cataract surgery. Recently, HA receptors have been found on liver endothelial cells that play an important role in HA catabolism. It is unknown if similar receptors are present on the corneal endothelium. In this study we have used two different methods to follow the interaction of HA with corneal endothelial cells: (1) binding of 3H-HA to cells or intact corneas was determined in the presence or absence of unlabelled glycosaminoglycans after solubilization with KOH, and (2) the HA-binding region of bovine cartilage proteoglycan was used as a histochemical probe and visualized by an avidin-biotin method. 3H-HA bound both to intact rat corneas pretreated with Streptomyces hyaluronidase and to cultured monkey corneal endothelial cells. The fraction-bound 3H-HA increased with time and was saturable. Cultured endothelial cells were estimated to have 1700-2100 binding sites per cell with a binding constant of 5.6-8.5 X 10(9) liters/mol. Furthermore, unlabelled HA displaced the tritiated in a dose-dependent manner and the displacing efficiency was dependent on molecular weight. The histochemical method disclosed that HA forms a continuous layer on the endothelium. If Healon was injected into the anterior chamber, the thickness and staining intensity of this layer increased conspicuously.

Accumulation of hyaluronic acid in the alveolar interstitial tissue in bleomycin-induced alveolitis.

By using biotin-labeled proteoglycan core protein and an avidin-enzyme system, hyaluronic acid (HA) was visualized in the lungs of rats at different times (4, 10, and 20 days) after bleomycin injury. Four days after an intratracheal injection of bleomycin, HA was accumulated in the edematous alveolar septa of the focal areas with lung tissue injury. An interstitial cellular infiltrate of mainly lymphocytes was present. In normal rat lung, HA was not seen in the alveolar tissue but confined to peribronchial and perivascular spaces. Ten and twenty days after bleomycin administration, increasing numbers of macrophages were apparent in the alveolar space. Proliferating fibroblasts and deposition of collagen in the alveolar tissue were observed while the diffuse HA accumulation was becoming less prominent in the alveolar interstitial tissue. HA was more distinctly located in the surroundings of proliferating fibroblasts. A few scattered alveolar macrophages showed a positive staining for HA. An increased water content of the lung was most apparent 4 days after bleomycin administration. The accumulation of HA, a glycosaminoglycan with unique qualities to immobilize water, in the alveolar interstitium suggests a role for HA in the alveolar interstitial edema. The appearance of HA in alveolar macrophages might indicate that macrophage phagocytosis contributes to the elimination of HA from inflamed lung tissue.

Carbonic anhydrase isoenzymes in the caecum and colon of normal and germ-free rats.

Histochemical and immunocytochemical methods were used to study the presence of carbonic anhydrase (CA) isoenzymes in the caecum and colon of normal and germ-free rats. Very high enzyme activity was demonstrated by histochemistry in the caecum and proximal colon of normal rats, while the activity decreased in the distal colon. Very strong immunostaining for the isoenzyme CA I was found in the cytoplasm of surface cells and upper gland cells in the caecum and colon of normal rats. In the distal colon the staining was less intense with a marked cell-to-cell variation. Ca II was found in the apical (luminal) cell region of the surface epithelium in all regions. Ca III was possibly present in small amounts, but this could not be judged with certainty. There was no difference in carbonic anhydrase between normal and germ-free rats (except for less staining of the mucosal capillaries in germ-free animals). Therefore, our data give no support to the hypothesis that CA I participates in the absorption of microbial fermentation products. The location of CA II in the apical cell region suggests a role for this isoenzyme in regulation of the microclimate close to the epithelial cells.

Carbonic anhydrase isoenzymes CA I and CA II in the human eye.

The distribution of carbonic anhydrase was studied in human donor eyes by the cobalt-phosphate histochemical method of Hansson and by immunofluorescence and immunoperoxidase techniques using antisera specific against the human cytoplasmic isoenzymes CA I and CA II. Corneal endothelium displayed specific immunological staining for CA I and CA II. Distinct enzyme activity was observed histochemically in the plasma membranes and cytoplasm of the endothelium. In the ciliary processes immunological evidence for the presence of CA II was found both in pigmented (PE) and in nonpigmented (NPE) epithelium. Activity was observed in the cytoplasm and basolateral membranes of NPE, but only in the basal membranes of PE. In the lens the plasma membranes of both the epithelium and fibers displayed intense activity, whereas cytoplasmic enzyme activity was seen only in the epithelium. There was no activity in the lens capsule. Immunofluorescence studies were difficult because of autofluorescence, but the immunoperoxidase technique indicated the presence of both CA I and CA II in the lens. In the central retina, Müller cells stained for CA II. Histochemically, enzyme activity was seen in the cytoplasm and at the plasma membranes. Activity was also observed in some but not all cones. Electron microscopy revealed this to be located in the cristae and plasma membranes adjacent to the pigment epithelium. Activity was also found in PE. Neurons and rods lacked both immunological staining and activity. Endothelial cells of capillaries in ciliary processes and in the choroid stained for CA I and exhibited histochemical activity, particularly those which faced neighboring epithelial cells containing the enzyme. The isoenzyme CA III, which is resistant to inhibition by sulfonamides, did not appear to be present in these ocular tissues, since the histochemical staining of enzyme activity was completely abolished by 10(-6) M acetazolamide.