Masatsugu Nakamura

Differential distribution of subchains of the basement membrane components type IV collagen and laminin among the amniotic membrane, cornea, and conjunctiva.

PURPOSE Amniotic membrane transplantation has been reported to be an effective surgical procedure for the reconstruction of the anterior segment of the eye. To understand better the function of transplanted amniotic membrane, we compared the distributions of subchains of type IV collagen and laminin in the amniotic membrane to those in the cornea and conjunctiva. METHODS Five human corneas with conjunctivas and three human amniotic membranes were frozen in OCT compound. Cryosections were cut with a cryostat and stained by an indirect immunofluorescent microscopy. We used antibodies of the collagen alpha2(IV) and alpha5(IV) subchains, laminin-1, laminin-5, fibronectin, and type VII collagen. RESULTS In the conjunctival basement membrane and the amniotic membrane, fluorescence was evident for collagen alpha2(IV) but not for collagen alpha5(IV). By contrast, in the corneal basement membrane, fluorescence was apparent for the collagen alpha5(IV) subchain but not for the collagen alpha2(IV) subchain. Laminin-1, laminin-5, fibronectin, and type VII collagen were present in all the basement membranes examined. CONCLUSION The distribution of alpha subchains of type IV collagen in the amniotic membrane was identical to that in the conjunctiva but different from that in the cornea. No difference in the distribution pattern of other basement membrane components was observed. These results demonstrate that the basement membrane of the amniotic membrane and the conjunctiva might share the same components; therefore, the amniotic membrane might be useful as a replacement for the basement membrane of the conjunctiva.

Hyaluronan stimulates corneal epithelial migration

Hyaluronan (hyaluronic acid), well-known for its viscoelastic properties, is also recognized as a biological signal to cells. Using organ cultures of the rabbit cornea, we investigated the effects of hyaluronan on the migration of corneal epithelium. The addition of hyaluronan to the culture medium increased the length of the path of the corneal epithelial layer in a dose-dependent fashion. Other glycosaminoglycans (chondroitin, chondroitin sulphate, keratan sulphate and heparan sulphate) were also tried, but only hyaluronan exhibited a stimulatory effect on corneal epithelial migration. The effects of hyaluronan and fibronectin or epidermal growth factor (EGF) were additive; the addition of antisera against fibronectin or against EGF did not alter the stimulatory effect of hyaluronan. These results demonstrate that hyaluronan stimulates corneal epithelial migration by mechanism(s) different from those of fibronectin and EGF.

Synergistic effects of substance P with insulin-like growth factor-1 on epithelial migration of the cornea

We find that substance P (SP) and insulin‐like growth factor‐1 (IGF‐1) demonstrate a synergistic effect on the stimulation of rabbit corneal epithelial migration in an organ culture. The addition of either SP or IGF‐1 alone did not affect epithelial migration, while the combination of SP and IGF‐1 stimulated epithelial migration in a dose‐dependent fashion. The synergistic effects of SP and IGF‐1 on corneal epithelial migration were nulled by the addition of a SP antagonist or enkephalinase. Among neurotransmitters (vasoactive intestinal peptide, calcitonin gene‐related peptide, acethylcholine chloride, norepinephrine, serotonin) or tachykinins (neurokinin A, neurokinin B, kassinin, eledoisin, physalaemin), only SP demonstrated a synergistic effect with IGF‐1 on cellular migration. In contrast, the combination of SP and IGF‐1 did not affect the incorporation of 3H‐thymidine into corneal epithelial cells. The attachment of the corneal epithelial cells to fibronectin, collagen type IV, and laminin matrices increased after treatment of the cells with SP and IGF‐1, but SP or IGF‐1 by themselves did not affect the attachment of the cells to these extracellular matrix proteins. An identical synergistic effect on corneal epithelial migration was observed when an NK‐1 receptor agonist was used in place of SP, suggesting the synergistic effect of SP and IGF‐1 might be mediated through the NK‐1 receptor system. These results suggest that the maintenance of the normal integrity of the corneal epithelium might be regulated by both humoral and neural factors.

Artificial human corneas: Scaffolds for transplantation and host regeneration

Purpose To review the development of artificial corneas (pros-theses and tissue equivalents) for transplantation, and to provide recent updates on our tissue-engineered replacement corneas. Methods Modified natural polymers and synthetic polymers were screened for their potential to replace damaged portions of the human cornea or the entire corneal thickness. These polymers, combined with cells derived from each of the three main corneal layers or stem cells, were used to develop artificial corneas. Functional testing was performed in vitro. Trials of biocompatibility and immune and inflammatory reactions were performed by implanting the most promising polymers into rabbit corneas. Results Collagen-based biopolymers, combined with synthetic crosslinkers or copolymers, formed effective scaffolds for developing prototype artificial corneas that could be used as tissue replacements in the future. We have previously developed an artificial cornea that mimicked key morphologic and functional properties of the human cornea. The addition of synthetic polymers increased its toughness as it retained transparency and low light scattering, making the matrix scaffold more suitable for transplantation. These new composites were implanted into rabbits without causing any acute inflammation or immune response. We have also fabricated full-thickness composites that can be fully sutured. However, the long-term effects of these artificial corneas need to be evaluated. Conclusions Novel tissue-engineered corneas that comprise composites of natural and synthetic biopolymers together with corneal cell lines or stem cells will, in the future, replace portions of the cornea that are damaged. Our results provide a basis for the development of both implantable temporary and permanent corneal replacements.

INS365 Suppresses Loss of Corneal Epithelial Integrity by Secretion of Mucin-like Glycoprotein in a Rabbit Short-term Dry Eye Model

P2Y2 receptor agonists, like UTP and ATP, stimulate mucin secretion from goblet cells in vitro. Therefore, mucin stimulants could be good candidates for the treatment of dry eye syndrome because mucin increases the tear film stability and protects against desiccation of ocular surface. INS365 is a more stable P2Y2 receptor agonist than UTP. In the present study, we evaluated, in normal rabbit eyes, its effectiveness to release mucin from goblet cells and to protect the corneal damage induced by desiccation. For mucin secretion, impression cytology was performed following the instillation of INS365 solution or saline into the conjunctival sac. The specimens were stained with periodic acid and Schiff (PAS) reagent, and then the staining area was calculated using computer software. INS365 dose-dependently decreased the PAS staining area of conjunctival goblet cells from 2 to 15 min post-application. Furthermore, we utilized the rabbit short-term dry eye model to evaluate if INS365 eyedrops could protect against any of the damage produced by blockage of blinking with ocular speculum. INS365 significantly suppressed corneal damage at concentrations of more than 0.1% w/v. These results suggest that this P2Y2 agonist is a good candidate for the treatment of dry eye disease.

Characterization of water retentive properties of hyaluronan.

We examined the water retentive properties of hyaluronan because of its reported therapeutic effect in the treatment of dry eye. Hyaluronan dose dependently retarded water loss from a solution kept at constant temperature and humidity. Similarly, water loss was retarded when hyaluronan was placed atop an agar gel. These decreases in water loss were not related to changes in the molecular weight of hyaluronan. Unlike the in vitro models, the evaporation rate from the tears in normal subjects initially increased following the topical application of hyaluronan, and continued a higher rate than with the vehicle. These results suggest that hyaluronan enhances water retention on the corneal surface, and probably increases corneal wettability. Accordingly, hyaluronan eye drops may be useful in the treatment of dry eye.

Diquafosol Elicits Increases in Net Cl– Transport through P2Y2 Receptor Stimulation in Rabbit Conjunctiva

The purpose of the present study was to understand the mechanisms of action of diquafosol, a stable derivative of uridine 5′-triphosphate, on Cl– transport across the isolated rabbit conjunctiva. Rabbit conjunctivas were isolated and mounted in a modified Ussing chamber. Under short-circuit conditions, the effects were determined of mucosal (tear) side diquafosol application on the short-circuit current (Isc). Diquafosol rapidly and dose-dependently increased the Isc at concentrations ranging from 0.1 to 968 µM when added to the mucosal side of the conjunctiva. In the absence of the serosal Cl–, the Isc induced by 10 µM diquafosol was substantially reduced. On the contrary, in the absence of mucosal side Na+, the diquafosol-induced increases in Isc were unchanged. Following 45-min preincubation, the P2Y2 antagonist suramin inhibited the diquafosol-induced increases in the Isc whereas the P2Y1 antagonist pyridoxal-phosphate-6-azophenyl-2′4′-disulfonic acid had no effect. These studies suggest that diquafosol stimulates net Cl– secretion from the serosal to the mucosal side via stimulation of P2Y2 receptors in the rabbit conjunctiva.

Diquafosol Ophthalmic Solution for Dry Eye Treatment

IntroductionThere has been rapid progress in our understanding of dry eye pathogenesis, as well as the development of improved diagnostic clinical tests. Various types of dry eye treatment drugs have been developed. This review summarizes the basic and clinical research carried out in the development of diquafosol for ophthalmic use.ResultsDiquafosol is a dinucleotide, purinoreceptor P2Y2 receptor agonist. Basic pharmacological studies have shown that it acts on P2Y2 receptors at the ocular surface, to promote tear and mucin secretion via elevated intracellular Ca2+ concentrations. Diquafosol also improves tear and mucin secretion in experimental dry eye models. Based on the results of laboratory experiments, the authors conducted a series of clinical studies in patients with dry eye disease. Diquafosol was effective in the treatment of dry eye disease at an optimal dose of 3% six times a day. In comparison to commercially available 0.1% sodium hyaluronate ophthalmic solution, 3% diquafosol ophthalmic solution showed non-inferiority in improving corneal fluorescein staining scores and superiority in improving keratoconjunctival Rose Bengal staining scores.ConclusionsDiquafosol ophthalmic solution has a novel mechanism of action that is characterized by its stimulatory effects on tear and mucin secretion. This drug has the potential to be effective in patients with tear film instability and short break-up time type of dry eye, which are essential factors in dry eye pathogenesis.

Innervated human corneal equivalents as in vitro models for nerve-target cell interactions

A sensory nerve supply is crucial for optimal tissue function. However, the mechanisms for successful innervation and the signaling pathways between nerves and their target tissue are not fully understood. Engineered tissue substitutes can provide controllable environments in which to study tissue innervation. We have therefore engineered human corneal substitutes that promote nerve in‐growth in a pattern similar to in vivo re‐innervation. We demonstrate that these nerves (a) are morphologically equivalent to natural corneal nerves; (b) make appropriate contact with target cells; (c) can generate action potentials; (d) respond to chemical and physical stimuli; and (e) play an important role in the overall functioning of the bioengineered tissue. This model can be used for studying the more general topics of nerve ingrowth or regeneration and the interaction between nerves and their target cells and, more specifically, the role of nerves in corneal function. This model could also be used as an in vitro alternative to animals for safety and efficacy testing of chemicals and drugs.

Concentration and molecular weight dependency of rabbit corneal epithelial wound healing on hyaluronan

Hyaluronan (hyaluronic acid) is a high molecular weight viscoelastic polymer which has been postulated to enhance wound healing. We investigated the dose and molecular weight (9 x 10(4)-280 x 10(4) dependent effects of hyaluronan on the rate of migration of rabbit corneal epithelium in organ culture and on wound closure in vivo after debridement with n-heptanol. When corneal blocks were cultured with hyaluronan for 20 hours, distances of epithelial migration significantly increased over exposed stroma in proportion to hyaluronan concentration. However, there was no difference in the stimulatory action of hyaluronan on epithelial migration when corneal blocks were cultured at 1 mg/ml of hyaluronan irrespective of changes in the molecular weight range between 9 x 10(4) and 280 x 10(4). Glycosaminoglycans other than hyaluronan (chondroitin, chondroitin sulfate, keratan sulfate and heparan sulfate) failed to increase the epithelial migration. When hyaluronan eye drops were instilled after corneal epithelial removal with n-heptanol, hyaluronan stimulated wound closure in a dose-dependent manner, but its stimulatory efficacy was not dependent on molecular weight.