The molecular weight of hyaluronate in the aqueous humour and vitreous body of rabbit and cattle eyes

The molecular weight distribution of hyaluronate in the aqueous humour and vitreous body of rabbit and cattle has been determined by gel chromatography. The eluate from the column was monitored by a radioassay, whereby the molecular weight distribution of 15–20 μg samples could be analysed. Control experiments were carried out with radioactively-labelled hyaluronate added to bovine material to estimate the degradation of the polymer during handling of the ocular fluids. It was shown that in vitro degradation does not appreciably affect the results.The analyses show a considerable polydispersity of the hyaluronate preparations. There is also a marked variation in the degree of polymerization between the two species. Hyaluronate from rabbit vitreous has a weight-average molecular weight ($\text{M}{}_{\mathrm{<mtext>w</mtext>}}$) of 2–3 × 10⁶ while adult bovine vitreous displays a value of about 5–8 × 10⁵. The hyaluronate of bovine vitreous varies with age. In newborn calf, a value of 3 × 10⁶ was registered. This value dropped to about 5 × 10⁵ in old cattle.The hyaluronate in the aqueous humour of rabbit showed a considerably higher molecular weight than that of the vitreous indicating that part of the hyaluronate in the anterior segment originates elsewhere than the vitreous. The differences between hyaluronate from the aqueous humour of adult cattle and that from the vitreous were more complex. As with the rabbit, a relatively large proportion of hyaluronate in the aqueous humour was of high-molecular weight, but, in contrast, the aqueous humour also contained material which had a lower degree of polymerization than the hyaluronate in the vitreous. The proportion of high-molecular weight material in bovine aqueous humour seemed to be lower in the summer than in the winter.     

高糖诱导牛视网膜微血管周细胞β-catenin的表达

目的:探讨高糖培养牛视网膜微血管周细胞β-catenin表达。方法:原代培养周细胞,用免疫细胞化学和Westernblot方法检测高糖诱导的牛视网膜微血管周细胞β-catenin蛋白表达。结果:视网膜微血管周细胞在5mmol/LD-葡萄糖培养72h后,β-catenin免疫反应性主要表现在细胞质,在30mmol/LD-葡萄糖培养72h后,β-catenin免疫反应性显著增加,β-catenin免疫反应性主要表现在细胞核和细胞质;Westernblot发现高糖组周细胞培养48,72h后β-catenin蛋白活性和表达较对照组显着增加。结论:高糖诱导的视网膜微血管周细胞β-catenin表达和活性显著增加。     

Lens exopeptidases

Exopeptidases identified as dipeptidyl peptidases II and III were found in the bovine, human normal and human cataractous lenses. Arylaminopeptidase activities identified in extracts of these tissues were able to hydrolyze phenylalanine, leucine, methionine, lysine and alanine arylamidase substrates. Lysosomal DAP II and the cytosol DAP III were recognized by their marked preferences for the release of Lys-Ala from Lys-Ala-2-NNap (at pH 5·5) and Arg-Arg from Arg-Arg-2-NNap (at pH 9·0), respectively. DAP III in human lens extracts exhibited a pH optimum of 9·5 at 37°C. The K_m for the DAP III substrate was estimated to be 4·64 × 10^?5m for normal lens extracts, 2·74 × 10^?5m for human cataractous lens extracts, and 2·83 × 10^?5m for bovine lens extracts. Lens DAP III was strongly inhibited by PCMS, EDTA, and to a lesser degree by puromycin and DFP. Arylaminopeptidase activities (from human and bovine lenses) assayed on Leu-2-NNap were strongly inhibited by PCMS and puromycin. Negligible effects were obtained with EDTA and DFP. Only traces of leucyl aminopeptidase (assayed on leucinamide) could be detected in human lenses; however, aminopeptidase activities measured at pH 7·0 on the β-naphthylamides of leucine, methionine, lysine, phenylalanine and alanine were plentiful in both human and bovine lenses, and served to indicate the possible presence of other aminopeptidases such as alanine aminopeptidase and aminopeptidase B. Of particular interest was the finding that the specific activities of Met-, Lys-, and Ala-2-NNap were 2–5-fold higher in cataractous human lenses compared to normal lenses.     

玻璃酸钠和重组牛bFGF滴眼液在角膜铁锈异物取出术后的疗效

目的:观察比较玻璃酸钠滴眼液和重组牛碱性成纤维细胞生长因子滴眼液联合应用对角膜铁锈异物取出术后角膜创面上皮修复的临床效果。方法:选择角膜铁锈异物患者98例98眼,随机分成联合治疗组49例49眼和对照组49例49眼。联合治疗组于角膜异物取出术后滴玻璃酸钠滴眼液+重组牛碱性成纤维细胞生长因子滴眼液+盐酸左氧氟沙星滴眼液;对照组滴重组牛碱性成纤维细胞生长因子滴眼液+盐酸左氧氟沙星滴眼液。每周3次观察角膜荧光素染色、角膜创面上皮修复及患眼局部症状等指标,观察2wk。结果:联合治疗组的总有效率96%高于对照组的88%(P<0.05)。结论:玻璃酸钠滴眼液和重组牛碱性成纤维细胞生长因子滴眼液联合应用能明显促进角膜损伤后上皮修复速度,疗效确切,安全可靠。     

Experimental endothelial lesions by means of an ultrasound phacoemulsificator

The current study deals with experimental use of an ultrasound generator for phacoemulsification and its effect on bovine corneal endothelium. The extent of the endothelial defect was defined by staining cell borders and nuclei. Histological examination showed that endothelial cells on the edge of a defect had no nuclei and were partially detached from the Descemet's membrane in some cases. Quantitative determinations of endothelial cell defects demonstrated the effects of distance from the generator (1, 2, and 3 mm) as well as duration of exposure. At 2 and 3 mm, addition of sodium hyaluronate (Healon) to the incubation medium was associated with a statistically significant reduction in size of the endothelial defect.     

The effects of denatured sodium hyaluronate on the corneal endothelium in cats

In an in vivo cat model, wide-field specular microscopy and scanning electron microscopy were used to observe whether heat-denatured sodium hyaluronate causes cell damage to the corneal endothelium. The endothelial cell toxicities of various drugs, including 0.01% benzalkonium chloride, 0.2% chlorhexidine digluconate, and 0.5% lidocaine, mixed with intact or untreated sodium hyaluronate were also investigated. Neither heat-denatured nor intact or untreated sodium hyaluronate alone had any adverse effect on the corneal endothelium in cats, whereas 0.01% benzalkonium chloride and 0.2% chlorhexidine digluconate, mixed with sodium hyaluronate, caused substantial endothelial morphologic changes, which resulted in corneal edema. By comparison, 0.5% lidocaine with sodium hyaluronate was found to have minimal effect on the corneal endothelium in cats. These findings indicate that some chemical contaminant with sodium hyaluronate, not heat-denatured sodium hyaluronate, induces an immediate onset of pseudophakic bullous keratopathy.     

Sodium hyaluronate as an ophthalmic vehicle: some factors governing its effect on the ocular absorption of pilocarpine

Sodium hyaluronate, as an additive to aqueous ophthalmic formulations, has been claimed to increase the ocular contact time and, thereby, the drug bioavailability. In the present study, the effect of sodium hyaluronate on corneal residence time and drug absorption in rabbits was investigated. Addition of sodium hyaluronate (0.125%) to a 3H-pilocarpine HCl solution resulted in increased retention of radioactivity in tear fluid and a 2-fold increase in drug concentration in the cornea and aqueous humor. Further, the effects of concentration and molecular weight of sodium hyaluronate on the miosis induced by pilocarpine in rabbits were studied. A significant increase of miotic response was seen at concentrations just less than 0.1% sodium hyaluronate. Pilocarpine solutions prepared from high mol.wt. sodium hyaluronate exhibited a greater miotic response than those prepared from lower mol.wt. samples. This might indicate that other physicochemical properties of sodium hyaluronate influence drug bioavailability.     

Evaluation of ophthalmic visicoelastic devices in rabbit penetrating keratoplasty--the protective effect for corneal endothelial cells after autokeratoplasty and the effect of intraocular pressure after aqueous exchange

PURPOSE: To investigate the effect of sodium hyaluronate molecular weight on corneal endothelial cells and intraocular pressure after penetrating keratoplasty in rabbits. MATERIAL AND METHOD: Rotating autokeratoplasty was carried out on rabbits using six groups of sodium hyaluronate in which the molecular weight differed. Then the endothelial cell density, hexagonal cell rate, corneal thickness, and intraocular pressure were measured. The intraocular pressure in the rabbit eyes after aqueous exchange with six groups of sodium hyaluronate was also measured. RESULTS: The endothelial cell density and hexagonal cell rate in the hyaluronate group of molecular weight 1.53-2.13 million and 1.9-3.9 million were the same as in the eye groups receiving no manipulation. As for the intraocular pressure after aqueous exchange, the highest intraocular pressure was more than 40 mmHg in the group treated with hyaluronate over 1.9 million and the group treated with hyaluronate and chondroitin sulfate compounding agent. CONCLUSION: Endothelial protection was considered to be superior when hyaluronate with a molecular weight of 1.5-3.9 million was used in rabbits. However, it is grossible that leaving a large amount of hyaluronate with a molecular weight over 1.9 million in the eye may lead to a remarkable increase in the early postoperative intraocular pressure.     

Influence of sodium hyaluronate on the meiotic effect of pilocarpine in rabbits

Sodium hyaluronate is a potential vehicle for drugs given topically to the eye. In the present study, the effect of sodium hyaluronate on the miosis induced by pilocarpine in rabbits was investigated. Addition of 0.2 and 0.75% sodium hyaluronate to 1% pilocarpine hydrochloride in buffer resulted in an increased effect measured as area under the miosis-time curve (AUC) and duration of miosis. A comparison between a commercially available 1% pilocarpine preparation and the preparations containing sodium hyaluronate showed that sodium hyaluronate improves the effect parameters. No adverse effects with sodium hyaluronate were observed. Possible mechanisms inducing these improvements are discussed.     

Biochemical studies on the use of sodium hyaluronate in the anterior eye segment. IV. The protective efficacy of the corneal endothelium

We devised an apparatus to add constant mechanical force to the corneal endothelium, and the protective efficacy of sodium hyaluronate to the corneal endothelium was determined quantitatively using an image analyzer. When the corneal endothelium was coated with a 1% solution of 800K or 2150K sodium hyaluronate, the damaged area was significantly less than cases having a coating with a 1% solution of 200K sodium hyaluronate or phosphate buffered saline which was the solvent of sodium hyaluronate. The water uptake of damaged cornea was also investigated using tritiated water. The cornea coated with 800K or 2150K sodium hyaluronate showed the same water uptake as the normal cornea, but the cornea coated with 200K sodium hyaluronate or phosphate buffered saline showed a significantly higher uptake. These results suggest that a 1% solution of 800K or 2150K sodium hyaluronate protects the corneal endothelium from mechanical damages.     

Conductivity of sodium hyaluronate solution]

In order to elucidate the property of water contained by the stereoic network in the vitreous gel, the conductivity of sodium hyaluronate solutions was measured in various concentrations at 25 degrees C and the relationship between concentrations of sodium hyaluronate and conductivity was evaluated. Each conductivity curve was divided into three stages. Equivalent conductivity was highly dependent upon the concentration of sodium hyaluronate at high and low concentrations, but was rather independent of it in intermediate concentrations. These results suggest that there are three types of water dependent upon the concentration of sodium hyaluronate, indicating physical variety, and that sodium hyaluronate solution in the intermediate concentration may possibly have a buffer homeostatic action on the ionic environment in the vitreous body.     

The influence of high molecular weight sodium hyaluronate (Healon) on the production of migration inhibitory factor

The effect of sodium hyaluronate on the production of migration inhibitory factor (MIF) was studied in a two step MIF-assay. High molecular weight sodium hyaluronate (100 micrograms/ml), added during the inductory step of the MIF-assay, inhibited the production of MIF. The inhibitory effect did not appear to be due to physical factors such as steric hindrance, which may prevent mitogen binding, since cells preactivated with phytohemagglutinin A (PHA) did not produce MIF when incubated in the presence of sodium hyaluronate. The inhibitory effect was still measurable when the sodium hyaluronate was added upto two hours after stimulation of the mononuclear cells with PHA. Inhibition was also found when the cells were preincubated with sodium hyaluronate, and washed prior to mitogen stimulation. Sodium hyaluronate could only be removed from the cells by incubation with hyaluronidase or by incubation of the cells for at least two hours in culture medium, whereafter the cells could be stimulated to the same extent as normal untreated cells to produce MIF. This inhibitory effect on cytokine production may explain the reduced inflammatory reactions found both in vivo and in vitro in the presence of sodium hyaluronate.     

Postoperative pressures after phacoemulsification: sodium hyaluronate vs. sodium chondroitin sulfate-sodium hyaluronate

This randomized study of 100 patients compared the effects of two viscoelastic agents on intraocular pressure after phacoemulsification cataract extraction with intraocular lens implantation. In 50 patients, sodium hyaluronate was instilled in the anterior chamber before lens insertion and then evacuated. Sodium chondroitin sulfate-sodium hyaluronate was instilled in another 50 patients and was not evacuated. Postoperative intraocular pressure increases occurred much less frequently (6% versus 54%) and were milder in the group that received sodium chondroitin sulfate-sodium hyaluronate. Mean peak intraocular pressure was reached at 16 hours in both groups; peak pressure was 31.7% higher in the group that received sodium hyaluronate. Better clearance from the eye due to the lower molecular weight of sodium chondroitin sulfate-sodium hyaluronate may explain the differences in intraocular pressures between the groups.     

Preparation of gels from hyaluronate solutions

Stable, transparent gels can be prepared from hyaluronate solutions by adding CuSO₄. The best gelation was found using solutions of 2 mg/ml hyaluronate in glass-distilled water at a pH of 6.2 after the addition of 1 mg/ml CuSO₄. Methylation of the carboxyl groups of the hyaluronate completely abolished the gelation, indicating the importance of the carboxyl groups for the gel formation with Cu²⁺ ions. Gelation also depends on the molecular size of the hyaluronate, since hyaluronate was not able to form a gel after depolymerization with hyaluronidase.     

人工晶体植入后房水中透明质酸钠残留量的测定

目的测定人工晶体植入后房水中透明质酸钠的残留量。方法用岛津ＵＶ３１０１型紫外分光光度仪测量２３例（２５只眼）人工晶体植入后,注吸前及注吸２ｍｌ、５ｍｌ、８ｍｌ时房水中透明质酸钠（ｓｏｄｉｕｍｈｙａｌｕｒｏｎａｔｅ）的百分比浓度。结果注吸前、后不同的相对百分比浓度分别为６５５０％±１８１２％、１０２０％±４８０％、２１９％±１８３％及０７０％±０９１％。结论房水置换２ｍｌ可清除房水中大部分透明质酸钠,而置换８ｍｌ可使透明质酸钠残留减至微量,保持术后眼压正常。     

Management of severe hypotony with intravitreal injection of viscoelastic

PURPOSE: To investigate the effect of an intravitreal injection of sodium hyaluronate 1.4% and 2.3% in the treatment of hypotony. DESIGN: Interventional case series. METHODS: Intravitreal injection of sodium hyaluronate 1.4% was performed to treat persistent hypotony in three eyes after the successful repair of retinal detachment and in one eye with uveitis. Injection of sodium hyaluronate 2.3% was performed in one eye after the successful repair of retinal detachment with removal of silicone oil. RESULTS: At the last follow-up examination (2-16 months), intraocular pressure increased to at least 5 mm Hg (5-14 mm Hg) in all eyes that had been injected with sodium hyaluronate. Intraocular pressure increased to 38 mm Hg in the eye that had been injected with sodium hyaluronate 2.3% on postoperative day 1 and was 10 mm Hg at three months. Vision improved in all patients. CONCLUSION: Intraocular injection of sodium hyaluronate 1.4 and 2.3% appears to be efficacious in reversing hypotony in some eyes. Functional and structural findings can also improve.     

挫伤性虹膜根部断离修复术中玻璃酸钠的应用

目的：观察玻璃酸钠在挫伤性虹膜根部断离显微修复术中应用的临床效果。方法：使用玻璃酸钠复位虹膜，运用显微手术技术修复虹膜根部断离。结果：本组7例都恢复了虹膜的解剖位置和瞳孔形态，视功能得到明显改善。结论：失伤性虹膜根部断离显微修复术中应用玻璃酸钠是理想的手术方法。     

Effect of 1% sodium hyaluronate (Healon) on a nonregenerating (feline) corneal endothelium

A series of experiments were performed to investigate the effect of 1% sodium hyaluronate (Healon) on the nonregenerating corneal endothelium of the cat. Aqueous humor replacement with 1% sodium hyaluronate resulted in mild, transient elevations of intraocular pressure compared to eyes that were injected with balanced salt solution. Sodium hyaluronate 1% protected the feline endothelium against cell loss incurred by contact with hyaluronate-coated intraocular lenses compared to endothelial contact with lenses that were not coated with sodium hyaluronate. The use of intraoperative 1% sodium hyaluronate, however, did not protect against endothelial cell loss incurred by penetrating keratoplasty or prevent subsequent skin graft-induced corneal homograft rejections. Homograft rejections were milder, however, in some eyes that received grafts coated with 1% sodium hyaluronate. Image analysis of photographs of trypan blue- and alizarin red-stained corneal buttons after trephining, stretching of Descemet's membrane, rubbing against iris-lens preparations, or immediately after penetrating keratoplasty demonstrated that the stretching of the posterior cornea is an important cause of endothelial damage that would not be protected against by a viscoelastic coating.     

Scheimpflug photography study of ophthalmic viscosurgical devices during simulated cataract surgery

PURPOSE: To evaluate by Scheimpflug photography the capacity of several commercially available ophthalmic viscosurgical devices (OVDs) to remain in the anterior chamber and maintain anterior chamber depth (ACD) during simulated cataract surgery in porcine cadaver eyes. SETTING: Department of Preclinical Ophthalmology, Pharmacia & Upjohn AB, Uppsala, Sweden. METHODS: Eighty eyes from newly slaughtered pigs were used. They were distributed equally among 8 OVDs: Amvisc Plus (sodium hyaluronate 1.6%), Biolon (sodium hyaluronate 1.0%), Biolon Prime (sodium hyaluronate 1.2%), Healon (sodium hyaluronate 1.0%), Healon GV (sodium hyaluronate 1.4%), Healon5 (sodium hyaluronate 2.3%), Provisc (sodium hyaluronate 1.0%), and Viscoat (sodium hyaluronate 3.0%-chondroitin sulfate 4.0%). Scheimpflug photographs were taken using a Nidek EAS-1000 instrument before surgery (control), after injection of the OVD, after continuous curvilinear capsulorhexis (CCC), and after lens extraction by phacoemulsification. Differences in the ACD with a P value less than 0.05 were considered statistically significant. The retention capacity was visually assessed. RESULTS: Healon5 had a significantly higher capacity to maintain the ACD than the other OVDs after CCC and phacoemulsification. After injection, Healon5, Viscoat, and Biolon Prime showed similar ACD-maintaining capacities. CONCLUSIONS: The capacity of Healon5 to maintain the ACD during cataract surgery, ie, neutralizing the vitreous pressure and stabilizing the anterior segment of the eye, was significantly higher than that of the other OVDs.     

Aggregation of deamidated human βB2-crystallin and incomplete rescue by α-crystallin chaperone

Aging of the lens is accompanied by extensive deamidation of the lens specific proteins, the crystallins. Deamidated crystallins are increased in the insoluble proteins and may contribute to cataracts. Deamidation has been shown in vitro to alter the structure and decrease the stability of human lens βB1, βB2 and βA3-crystallin. Of particular interest, βB2 mutants were constructed to mimic the effect of in vivo deamidations at the interacting interface between domains, at Q70 in the N terminal domain and at Q162, its C-terminal homologue. The double mutant was also constructed. We previously reported that deamidation at the critical interface sites decreased stability, while preserving the dimeric 3D structure. In the present study, dynamic light scattering, differential scanning calorimetry and small angle X-ray scattering were used to investigate the effect of deamidation on stability, thermal unfolding and aggregation. The bovine βLb fraction was used for comparative analysis. The chaperone requirements of the various samples were determined using bovine α-crystallins as the chaperone. Deamidation at both interface Gln residues or at Q70, but not Q162, significantly lowered the temperature for unfolding and aggregation, which was rapidly followed by precipitation. This deamidation-induced aggregation and precipitation was not completely prevented by α-crystallin chaperone. A potential mechanism for cataract formation in vivo involving accumulation of deamidated β-crystallin aggregates is discussed.