Lysosomal enzymes in corneal storage media and corneal graft outcome.

PURPOSE: The purpose of this study was to relate lysosomal enzyme activities in corneal storage media to the outcome of the transplanted corneas. METHODS: Corneal storage media from 358 transplanted corneas were frozen at -70 degrees C and kept for enzyme analysis. Corneas were stored in K-Sol (28), CSM (35), Dexsol (80), Index medium (five), Optisol (158), and Optisol GS (52). Activities of alpha-D-mannosidase, beta-glucuronidase, alpha-glucosidase, and N-acetyl-beta-glucosaminidase were assayed fluorometrically. Mayo Clinic records were examined for donor information, including cause of death and 2-month graft follow-up data. RESULTS: For all corneas, there was a low but significant correlation between activities of each enzyme and storage time (rs = 0.13-0.35; p = 0.02-0.0001), and donor age (rs = -0.14 to -0.23; p = 0.009-0.0001). There was no significant correlation of enzyme activity with 2-month endothelial cell density, structure, cell loss, or corneal thickness. Enzyme activities for four primary donor failures and six grafts with > 65% 2-month endothelial cell loss were not significantly different from those for the rest of the transplanted corneas. Enzyme activities were higher for corneas from donors with renal failure but not from those with diabetes mellitus. There was no significant difference in graft outcome for different cause-of-death groups. CONCLUSIONS: The activities of lysosomal enzymes released into corneal storage media are not useful as predictors of graft outcome.

Effect of scleral buckling on unsutured cataract wound strength.

BACKGROUND AND OBJECTIVE: Modern cataract surgery has evolved to include small, unsutured wounds with rapid visual rehabilitation and fewer complications. Properly constructed, these unsutured wounds can withstand increased intraocular pressures without leakage, rupture, or incarceration of intraocular contents. However, scleral buckling surgery may alter the architecture of these wounds and thus their strength. The authors wanted to study the effect of scleral buckling on the integrity of these unsutured cataract wounds. MATERIALS AND METHODS: Eighteen fresh human globes underwent creation of scleral and limbal corneal incisions so as to create self-sealing wounds. Scleral buckles were then placed. Intraocular pressures were elevated to 400 mm Hg before and after placement of the scleral buckles and evidence for wound leakage was sought. RESULTS: Two globes with clear corneal incisions and no scleral buckles leaked slightly at 300 mm Hg, but no globe with a scleral buckle, regardless of incision type or silicone element style, leaked at pressures to 400 mm Hg. CONCLUSION: Sutureless cataract incisions, if properly constructed, provide a strong, pressure-resistant wound. Scleral buckling does not appear to affect the strength of these wounds.

Age-related changes of glycosidases in human retinal pigment epithelium.

This study was undertaken to determine whether there are age-related changes in the specific activities of several glycosidases in fresh retinal pigment epithelial cells (RPE) isolated from the posterior pole of human donor eyes. One hundred and twenty-one pairs of eyes from human donors, between the ages of 43 and 95 years, were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA) and the Cleveland Ohio Eye Bank within 18 to 24 h of death. None had histories of diabetes, hepatitis, HIV infection, intraocular surgery, or documented age-related macular degeneration, although several older donors with evidence of drusen were included in the study. RPE cells were isolated from the posterior third of the retina using the conventional rush method and homogenized with a glass, Broeck tissue grinder. All post-nuclear supernatants were analyzed for glycosidase activity; a smaller number of nuclear pellets were assayed to verify that the majority of the enzyme activity was associated with the post-nuclear sypernatants. Glycosidase activity was quantitated fluorometrically by measuring the enzymatic release of umbelliferone from synthetic substrate preparations, specific for each enzyme. Total protein was determined by a micro BCA protein assay. Regression analysis revealed statistically significant age-related decreases for the specific activities of alpha-mannosidase (p = 0.0001), beta-galactosidase (p = 0.0001), N-acetyl-beta-glucosaminidase (p = 0.0001), and N-acetyl beta galactosaminidase (p = 0.0001) in fresh human donor RPE cells taken from the region of the posterior third of the retina that included the macula. Mannose and N-acetyl-glucosamine are major carbohydrate monomers of the oligosaccaride chains of human rhodopsin, and a relatively high percentage of the oligosaccharide chains are galactosylated. Defects in their degradation may lead to the accumulation of undigested residual material in the RPE.

The effect of hEGF and insulin on corneal metabolism during Optisol storage.

We examined the effect of the growth factors, human epidermal growth factor (hEGF) and insulin, on corneal metabolism during storage in Optisol, a chondroitin-sulfate-(CS)-based storage medium. Paired cat corneas, in either Optisol only or Optisol with growth factor(s), were analyzed using ex vivo 31P nuclear magnetic resonance, after storage for 1 week at 4 degrees C. Lysosomal enzyme release into the media at the end of the storage period also was measured fluorometrically. Both epithelial-intact and epithelial-denuded corneal pairs were examined for all conditions. Considering corneas having either intact epithelia or epithelium-denuded corneas, the addition of either growth factor alone to Optisol did not alter the relative corneal concentrations of five of the six phosphatic metabolite spectral bands measured or two metabolic ratios calculated from these bands. Phosphodiesters, however, were significantly lower in corneas stored in Optisol containing both hEGF and insulin (23%) than in corneas stored in Optisol alone (30%). Intracorneal pH was unaffected by the addition of growth factor(s). A significantly higher release of alpha-glucosidase and alpha-mannosidase was noted in those corneas stored in Optisol containing both hEGF and insulin. Optisol maintains high-energy phosphate corneal metabolism similar to other CS-based media, K-Sol and Chondroitin Sulfate Corneal Storage Medium (CSM). The addition of the growth factors hEGF and insulin to Optisol alters corneal metabolic activity during storage in a manner indicative of conserving corneal phospholipids.

A randomized, prospective, double-masked clinical trial of Optisol vs DexSol corneal storage media.

We compared Optisol and DexSol, two chondroitin-sulfate-based media for corneal storage at 4 degrees C, by transplanting 31 donor cornea pairs (one cornea stored in Optisol and its mate in DexSol for 20 to 134 hours) into 31 pairs of recipients (62 patients). All grafts were clear 1 year after transplantation except for one primary donor failure (Optisol group). Optisol-stored corneas were significantly thinner than DexSol-stored corneas after cardinal suture placement (0.64 mm vs 0.76 mm) and at the end of surgery (0.69 mm vs 0.78 mm); at all points afterward through 1 year the two groups did not differ. The activity of two lysosomal enzymes released into the media during storage, alpha-mannosidase and alpha-glucosidase, was lower in the Optisol group. Epithelial status and all endothelial morphometric parameters, except the figure coefficient at 1 year, did not differ between the two groups before surgery and 3, 6, and 12 months after surgery. Mean endothelial cell loss at 1 year was 15% for the Optisol group and 21% for the DexSol group (P = .22). Thus, Optisol-stored corneas were thinner during surgery than DexSol-stored corneas and there was less lysosomal enzyme activity in the Optisol medium after tissue storage. There were no significant differences in postoperative clinical or endothelial morphometric parameters, however.

An in vitro and clinical comparison of corneal storage with chondroitin sulfate corneal storage medium with and without dextran.

The safety and efficacy of 1% dextran in Chondroitin Sulfate Corneal Storage Medium (CSM) in reducing corneal swelling after 4 degrees C storage was assessed in a corneal endothelial cell culture system. No difference was found in 3H-thymidine incorporation by cells incubated in either CSM-dextran medium or CSM medium alone. Subsequently, 21 pairs of corneas, stored in either CSM or CSM-dextran from 30 to 112 hours, were transplanted into 42 eyes of 42 patients, paired by diagnostic group and procedure. All CSM grafts and 19 of 21 CSM-dextran grafts were clear at 4 months with no primary donor failures in either group. Intraoperative corneal thickness was significantly greater in the CSM group (0.82 +/- 0.07 mm) than the CSM-dextran group (0.76 +/- 0.06 mm); however, the two groups did not differ thereafter. No differences in all endothelial morphometric parameters were noted between the two groups pre- and postoperatively. Average endothelial cell loss by 4 months was 13.0 +/- 16.4% for the CSM group and 16.4 +/- 15.5% for the CSM-dextran group. The addition of dextran to CSM medium results in significant intraoperative corneal thinning without adversely affecting endothelial DNA synthesis in vitro and endothelial survival in vivo.

A donor-age-dependent change in the activity of alpha-mannosidase in human cultured RPE cells.

Six acidic glycosidase activities in cultured human retinal pigment epithelium (RPE) cells from donors of different ages (19 to 80 years) were studied with regard to pH optimum, Km, Vmax and specific activity. For alpha-mannosidase we found significant age-dependent decreases in specific activity and Vmax but not in Km. The other glycosidases and acid phosphatase, lactate dehydrogenase (LDH) and citrate synthase showed no change in these parameters with donor age. The alpha mannosidase activity of older donor cells could be activated almost 2-fold by the addition of zinc. This is the first report of age-dependent change in a human RPE lysosomal enzyme. Since alpha-mannosidase is probably required for the degradation of rhodopsin in the phagolysosomal system of the RPE, decrease in this enzyme activity may lead to accumulation of undigested rod outer segments (ROS) and drusen, both of which are associated with age-related macular degeneration (AMD).

Comparison of corneal storage in K-Sol and chondroitin sulfate corneal storage medium in human corneal transplantation.

Fifty-one pairs of corneas, stored in either K-Sol or CSM (chondroitin sulfate corneal storage medium) from 8 to 97 hours (mean +/- standard deviation, 58 +/- 21 and 57 +/- 21 hours, respectively), were transplanted in a prospective, randomized manner into 99 patients (n = 102 eyes), paired by diagnostic group and procedure. Ninety-six percent of K-Sol grafts (n = 51) and 94% of the CSM grafts (n = 51) were clear at 6 months; 92% of both the K-Sol (n = 38) and CSM (n = 35) grafts were clear at 12 months. One primary donor failure occurred, a K-Sol cornea stored for 76 hours. The CSM group experienced a greater number of persistent epithelial defects beyond 2 weeks (7 versus 4 defects) and graft reaction episodes (7 versus 3 episodes) than the K-Sol group; however, an equal number of late graft failures (3) occurred in both groups. No significant differences by paired t test analyses were found in endothelial cell density, area, coefficient of variation, or figure coefficient at 3 (n = 37 pairs), 6 (n = 36 pairs), and 12 (n = 26 pairs) months between the two groups. Mean endothelial cell density significantly decreased by 11% +/- 22 by 3 months in the K-Sol group, whereas the 7% +/- 24 decrease in the CSM group was insignificant. By 12 months, both groups experienced a significant decrease: K-Sol, 27% +/- 22; CSM, 17% +/- 26. A significant decrease in the mean coefficient of variation (polymegathism) was noted after 3 months in the K-Sol group which returned to the preoperative mean by 1 year, whereas this parameter remained unchanged in the CSM group. Both chondroitin sulfate-based media result in successful corneal transplantation with storage up to 4 days; however, endothelial survival with both media are comparable with previous studies with McCarey-Kaufman (M-K) medium.

Endophthalmitis due to Propionibacterium acnes sequestered between IOL optic and posterior capsule.

A 68-year-old woman had delayed onset, persistent uveitis following routine extracapsular cataract extraction with posterior chamber intraocular lens implantation. The patient initially responded to topical steroids, but developed a whitish capsular plaque through to represent possible Propionibacterium acnes endophthalmitis. A vitrectomy and capsular biopsy yielded cultures positive for P. acnes only after nine days. The intraocular lens was left in place. Light and electron microscopy revealed bacteria sequestered within the capsular bag.

Sustained release of 5-fluorouracil from ethylene acetate copolymer.

Pharmacologic inhibition of fibroblast proliferation and scar formation remains an important consideration in many ophthalmologic surgical procedures. Such inhibition is particularly important in glaucoma filtration surgery. A sustained release system for 5-fluorouracil (5-FU) using ethylene vinyl acetate copolymer (Elvax) was characterized in vitro. 5-FU was incorporated into Elvax discs of 4 mm diameter. Discs were coated to allow sustained and predictable release over a 14 day period. Alteration of coatings, which act as rate-controlling membranes, allowed variation in the pattern of 5-FU release from the core which acts as the drug reservoir. This system for the sustained release of 5-FU may prove valuable in glaucoma surgery where the suppression of cellular proliferation is desired. The non-inflammatory nature of Elvax suggests this compound may provide an alternative means of 5-FU delivery.

Lysosomal enzyme levels in corneal storage media. K-Sol versus McCarey-Kaufman.

Evidence indicates that lysosomal enzymes can carry out corneal autolysis during corneal storage and that they are damaging to the corneal endothelium. The authors investigated the release of lysosomal enzymes into two corneal storage media (K-Sol and McCarey-Kaufman [M-K]) by paired human donor corneas during 4 degrees C storage. The authors also studied the interaction of these media with lysosomal enzymes from human cornea. K-Sol and M-K stimulated (P less than 0.01) both beta-glucuronidase and alpha-galactosidase about equally. beta-N-Acetyl-glucosaminidase, a major catabolic enzyme of the cornea, was inhibited by the chondroitin sulfate in K-Sol by over 90% (P less than 0.01). Corneas stored in M-K released more lysosomal enzymes than corneas stored in K-Sol. At 4 days, the values approached significance (P less than 0.06) and by day 10 significantly higher values were found in the M-K media (P less than 0.01). Both storage methods showed a linear release. Individual corneas were found to vary in their release rates. Whether corneas that release more enzyme will show higher endothelial cell loss or produce less successful penetrating keratoplasty grafts deserves further study.

Sex chromosome aneuploidy and Bardet-Biedl syndrome.

The inheritance of Bardet-Biedl syndrome is thought to be autosomal recessive. Of the approximately 500 case reports in the literature, three patients were found to have sex chromosome aneuploidy. The authors describe two siblings with Bardet-Biedl syndrome, one of whom has a unique sex chromosome aneuploidy with mosaicism, including deletion of the short arm of the X chromosome (45,X/46,X,del(X)(p21)). The possible significance of sex chromosome aneuploidy and the Bardet-Biedl syndrome is discussed.

Internal sclerectomy for glaucoma filtering surgery with an automated trephine.

A technique is described for performing full-thickness glaucoma filtering operation from within the anterior chamber. The procedure utilizes an automated trephine, designated the "trabecuphine," that is capable of simultaneous cutting and irrigation. The trabecuphine is brought into the anterior chamber through a limbal incision 180 degrees away from the planned filter site. The trephine is passed across the anterior chamber, above the iris, and into the angle to perform the sclerotomy. This procedure was performed successfully in 13 eyes of seven cynomolgus monkeys. Complications included two small hyphemas that cleared completely. Other complications such as flat anterior chamber, corneal damage, iridodialysis, conjunctival buttonhole, and cataract formation did not occur. Internal scleral trephination may offer another alternative in the surgical management of patients with intractable glaucoma.

A case of epithelial ingrowth primarily involving the lens capsule.

We describe an unusual case of epithelial ingrowth primarily involving the lens capsule. Four years following removal of the lens and epithelial tissue the eye was quiet with 20/20 acuity. Histopathology showed intraocular epithelium attached to the anterior surface of the lens capsule.

Beta-N-acetyl-glucosaminidase: possible role in ocular neovascularization.

The possibility that lysosomal enzymes might be involved as angiogenic factors in ocular neovascularization (NV) was investigated. Beta-N-acetyl-glucosaminidase (NAGase) activity, and that of two other glycosidases, were present in the retinal derived protein fraction (RDPF) reported by others to be angiogenic. NAGase, but not the other glycosidases, was inhibited by vitreous. NAGase exhibited the same stability characteristics as RDPF. In diabetic rats there was a significant rise in vitreous but a fall in retinal NAGase activity. The sera of these animals, however, showed elevation in the activities of all five glycosidases. Preliminary experiments indicate that only the intermediate isoenzyme of NAGase, putatively insulin dependent, is elevated in the eyes of these diabetic rats. NAGase was also specifically elevated in the intraocular fluid from monkey eyes with retinal vein occlusion (RVO), and markedly so if NV was present. These results suggest the involvement of NAGase in the neovascular process in the eye.

Corneal alpha-galactosidase deficiency in macular corneal dystrophy.

Glycosidases, which cleave sugar molecules from complex glycopolymers, have been previously quantified in normal human cornea in our laboratory. Data quantifying glycosidases in macular corneal dystrophy are lacking. Tissue obtained at keratoplasty from patients with macular dystrophy and normal corneas obtained from eye bank eyes were used to determine levels of glycosidase activity. A fluorometric technique was employed using 4-methyl-umbelliferyl-glycosides as substrates. The corneal tissues were homogenized, centrifuged, and the supernatants assayed for enzyme activity. Specific activities (mumol/mg protein/hour) were determined and Km and Vmax values were obtained for all but one enzyme. Activity of alpha-galactosidase was significantly lower in cornea tissue and keratocytes from macular corneal dystrophy compared to normal.

Acquired fat hernia of the eye and orbit.

A case of bilateral acquired fat hernia of the eyes and orbit occurred in an obese male. The differential diagnosis and treatment are discussed.