Oxidative metabolism of 1-(2-chloroethyl)-3-alkyl-3- (methylcarbamoyl)triazenes: formation of chloroacetaldehyde and relevance to biological activity.

(Methylcarbamoyl)triazenes have been shown to be effective cancer chemotherapeutic agents in a number of biological systems. Because of their chemical stability, it is likely that their activity in vivo is the result of a metabolic activation process. Previous studies have shown that 1-(2-chloroethyl)-3-methyl-3-(methylcarbamoyl)triazene (CMM) and 1-(2-chloroethyl)-3-benzyl-3-(methylcarbamoyl)triazene (CBzM) are metabolized by rat liver microsomes in the presence of NADPH to yield the ((hydroxymethyl)carbamoyl)triazene analogs of the parent compounds. The present studies show that both compounds are also oxidized at the chloroethyl substituent to yield chloroacetaldehyde and a substituted urea. In the case of CBzM metabolism, 47% of the metabolized parent compound was recovered as benzylmethylurea, 8% was recovered as benzylurea, and 26% was recovered as the ((hydroxymethyl)carbamoyl)-triazene and carbamoyltriazene metabolites. These results suggest that the chloroethyl group is the favored initial site of metabolism. In reaction mixtures containing initial concentrations of 300 microM CBzM, 78 microM chloroacetaldehyde was produced, as compared to 58 microM chloroacetaldehyde produced from the metabolism of 300 microM CMM. The formation of chloroacetaldehyde, a known mutagenic DNA alkylating agent, may explain the biological activity of these compounds.

Transplantation of corneal endothelial cells using a cell carrier device.

Penetrating keratoplasty is currently the only treatment for corneal endothelial dysfunction. Although corneal transplantation has a high success rate, a few problems still remain, such as the limited availability of donor grafts, the change in refraction after penetrating keratoplasty, and the higher chance of immune rejection. In this study, a coated hydrogel lens (Chiron Ophthalmics Inc., Irvine CA, U.S.A.) has been used as a carrier to transplant cultured homologous kitten and rabbit corneal endothelial cells into adult cats and rabbits. The transplantation procedure was the same in both species. Corneal endothelial cells from homologous rabbits or cats were seeded on coated hydrogel lenses and cultured until they reached a complete monolayer with an average cell density of 2,500 cells/mm2. Five weeks before transplantation surgery, corneal endothelial cells were scraped to induce corneal edema. The cell carrier device was then transplanted as follows: a trephine cut (7.7 mm) was made into the stroma, producing an outer corneal plug. The inner cornea was then cut by using a 5.5-mm trephine, and this inner plug was discarded. The implant was inserted and the outer corneal plug was sutured back into place. Corneas cleared completely within 3 days in both rabbits and cats, and stayed clear for an average of 40 days in rabbits and 50 days in cats. The histopathological evaluation of the rejected grafts showed vascularized retrocorneal membrane formation in cats, whereas in rabbits severe cellular infiltration of the stroma with neovascularization occurred without retrocorneal membrane formation.

Corneal endothelial cell transplantation using Descemet's membrane as a carrier.

Rabbit corneal endothelial cells were transplanted into the right eyes of four New Zealand white rabbits using bovine Descemet's membrane as a cell carrier. Descemet's membranes were dissected from cow eyes, flattened on 36 mm culture dishes, and cut into discs with a 6 mm trephine. Rabbit corneal endothelial cells were seeded onto the discs and maintained in tissue culture conditions for seven days until a complete monolayer with a cell density of 3,000 cells/mm2 was formed. Before transplantation, corneal edema was induced in the host animals by an endothelial scrape wound, which removed the corneal endothelium. Five weeks later, the animals were prepared for transplantation. A corneal button was cut and placed on a dissection table so the host Descemet's membrane could be removed. The donor Descemet's membrane with a complete monolayer of rabbit corneal endothelial cells was placed on the stroma of the corneal button. To keep the donor membrane in place, the button was cauterized at three peripheral points and sutured back into the host eye. Prednisolone sodium phosphate eyedrops and dexamethasone eyedrops and ointment were applied twice daily during the post-operative period. All grafts stayed clear for a period of 12 to 17 weeks. This study shows the potential for using Descemet's membrane as a carrier for corneal endothelial cell transplantation.

Simultaneous penetrating keratoplasty, extracapsular cataract extraction, and intraocular lens implantation.

The postoperative results of 210 consecutive eyes in 177 patients who had simultaneous penetrating keratoplasty, extracapsular cataract extraction, and posterior chamber intraocular lens (IOL) implantation (triple procedure) were reviewed. All procedures were performed by one surgeon between January 1980 and December 1989. The most common diagnosis requiring a triple procedure was Fuchs' endothelial dystrophy. The remainder of the patients required the procedure for either corneal scarring, keratoconus, or Chandler's syndrome. Of the 210 grafts, 191 (91%) remained clear with a mean follow-up of 53 months (range 6 to 116 months). The mean preoperative keratometry (K) reading (n = 161) was 44.51/44.85 (SD 4.86/4.94), and the mean postoperative K reading (n = 111) was 44.29/43.50 (SD 3.83/3.63). The mean IOL power (n = 196) was + 20.87 diopters (D) (SD 2.86). The mean postoperative best visual acuity (n = 166) was 20/65 with a range from 20/20 to less than 20/400, although 61% had 20/50 acuity or better and 92% had 20/100 acuity or better. The mean spherical correction in 155 eyes was - 1.38 (SD 2.89), and the mean positive refractive cylinder was + 3.21 (SD 2.20). One hundred forty six of the 155 eyes with reported refractions (96%) had 6 D or less of refractive cylinder. Correlation of the recipient-donor disparity % and refractive cylinder was significant such that as disparity increased refractive cylinder increased (r = 0.197, P = .021). Correlation of the recipient-donor disparity % and keratometric cylinder indicated a similar trend but was not significant (r = 0.105, P = .310).

Suture adjustment for postkeratoplasty astigmatism.

Excessive corneal astigmatism following penetrating keratoplasty is a frequent problem. A technique that adjusts a single running 10-0 nylon suture after keratoplasty was used in this series of patients. The procedure requires a keratometer, slitlamp, topical anesthesia, and tying forceps. In 52 eyes, with an average of 10.0 diopters of keratometric astigmatism, we adjusted the suture to flatten the steep corneal axis. We were able to reduce astigmatism an average of 7.2 diopters and this remained stable three months post wound revision.

Cataract formation following corneal transplantation in eyes with Fuchs' endothelial dystrophy.

In a retrospective analysis, 78 eyes in 58 patients that underwent corneal transplantation for Fuchs' endothelial dystrophy were examined for the presence of cataracts. Follow-up ranged from 1 to 17 years, with an average of 6.62 years. Thirty-four (44.1%) of the eyes developed cataracts sufficient enough to require surgical removal. The mean time from transplant to cataract formation averaged 4.9 years. The average age at the time of transplant surgery in the eyes that did not develop cataracts was 55.6, and in those that developed cataracts, 60.3 (p = 0.049). Thirteen percent of the transplants lost transparency following cataract extraction. The incidence of postkeratoplasty cataract increased at age 50 and occurred in 75% of eyes grafted in patients 60 years of age or older.

Refractive versus keratometric astigmatism postkeratoplasty.

In patients who had undergone penetrating keratoplasty, we compared the relationship between keratometric astigmatism and actual refractive cylinder required for visual correction in 115 eyes postoperatively. The average time from surgery to refraction was 49.5 months and from surgery to keratometry 50.3 months. Sixty-five eyes retaining their running 10-0 nylon sutures had an average of 5.43 diopters of keratometric cylinder and 2.95 D of refractive cylinder. The difference was highly statistically significant (P less than .001). This significant difference was true in eyes with keratoconus and Fuchs' endothelial dystrophy. In 50 eyes with all sutures removed, the average keratometry reading revealed 4.27 D of astigmatism while the cylindrical refraction required in spectacles averaged 3.70 D. The difference was not statistically significant (P = .018). The significant difference between astigmatism as measured by keratometry and by refraction in eyes with the sutures still in place should be taken into consideration when using suture removal or adjusting techniques after keratoplasty.

An index for estimating probabilities of surgical success for cataract patients with retinal disease.

Patients with cataracts and coincident retinal disease are at increased risk for unsuccessful cataract surgery. We developed a method for estimating probabilities of surgical success for older cataract patients with retinal disease. Using data from a prospective study of cataract surgery outcomes, 36 patients with both cataracts and retinal disease were identified, 13 of whom attained surgical success with 20/40 or better vision 4 months after surgery. Age, presurgical Snellen letter acuity, location of retinal lesions, and presurgical visual function in everyday life (newspaper reading or television viewing) were all reliable correlates of surgical outcome. These data were analyzed using multiple logistic regression to develop a clinical index combining presurgical Snellen letter acuity, location of retinal lesions, and age to predict surgical success. The resulting index demonstrated 83% sensitivity and 85% specificity in sample data, correctly predicting 83% of patients (predictive value positive: 73%; predictive value negative: 90%).

Moderate Fuchs' endothelial dystrophy ATPase pump site density.

The Na+, K+-ATPase pump site density on corneal endothelial cells from Fuchs' endothelial dystrophy corneas has been shown to be drastically decreased in end-stage disease (McCartney et al, Invest Ophthalmol Vis Sci 28:1955, 1987) and significantly increased in the early stages (Geroski et al, Ophthalmology 92:759, 1985) as compared to normal endothelium. In order to provide values for corneas between these two extremes, eye bank corneas from donors with no evidence of corneal edema but with guttata across the extent of the cornea were processed for autoradiography as well as immunohistochemistry. Pump site density was increased compared to end-stage disease but was less than values reported for either functional tissue or early stage disease. Similarly, immunohistochemistry results showed the amount of Na+, K+-ATPase antibody localization to be increased in respect to end-stage disease, but reduced as compared to functional tissue. These results suggest that pump site density on endothelial cells affected with Fuchs' endothelial dystrophy follows a gradual decline towards end-stage disease values as opposed to a sudden sharp deterioration after an initial increase.

Effect of carbachol on postoperative intraocular pressure.

Carbachol instilled in 125 eyes at the time of extracapsular cataract extraction and posterior chamber lens insertion had a statistically significant effect on lowering the postoperative intraocular pressure (IOP) at 24 hours (average IOP 14.0 mm Hg) and at 72 hours (average IOP 11.6 mm Hg) (P less than .001). The percentage of patients with an acute IOP elevation at 24 hours was also significantly reduced: carbachol 10% versus control 33% (P = .001). Carbachol's prolonged lowering of postoperative IOP may increase the safety of cataract surgery, particularly in patients with compromised optic nerves.

Functional outcome one year following cataract surgery in elderly persons.

We conducted a prospective study of 164 patients aged 70 and over who had undergone cataract removal and intraocular lens implantation to determine variables which predicted change in physical function experienced by the patients one year after cataract surgery. Path analysis was used to estimate direct and indirect effects of ADL change. Direct effects were estimated for mental status (-.35), mental status change (.51), binocular visual disability (-.39), binocular visual disability change over one year (.51), and baseline ADL (.36), with 45% of variance explained. Age and baseline mental status had important indirect effects through mental status change, and presurgical visual disability had important indirect effects through both visual disability change and mental status change. In summary, change in level of function one year after cataract surgery was not explained solely by change in vision, but was conditional on baseline function and baseline mental status and mediated in part by change in mental status.

Corneal decompensation following acute angle-closure glaucoma.

Ten eyes of nine patients with endothelial dystrophy requiring iridectomy for acute angle-closure glaucoma developed corneal edema sufficient to require penetrating keratoplasty. Because of the proximity of iris to cornea, simultaneous lens extraction was carried out to prevent malignant glaucoma. Penetrating keratoplasty using techniques presented here provided improved visual acuity and control of glaucoma in all cases.

A clinical index for predicting visual acuity after cataract surgery.

We developed a clinical index for predicting postoperative visual acuity of cataract patients and cross-validated it using data from 182 patients aged 70 years and older. The index consisted of four statistically combined indicators: age, preoperative visual acuity, frequency of reading, and comorbidity. Validation of the index included comparisons to two standard technical instruments for measurement of retinal visual acuity. For the clinical index, 72% of predictions were accurate within one Snellen line of postoperative visual acuity compared to 37% using a laser interferometer and 33% using a potential acuity meter. Testing of the clinical index's external validity using data from 111 patients in a different ophthalmology clinic disclosed 61% of predictions accurate within one Snellen line.

Surgery for corneal epithelial basement membrane dystrophy.

Seventeen consecutive eyes with corneal epithelial basement membrane with symptoms of recurrent erosion and/or decreased vision were treated with corneal epithelial basement membranectomy. Younger patients tended to have recurrent erosion, whereas older patients had decreased vision. All patients' symptoms improved following surgery. Electron microscopy revealed reduplication of the corneal epithelial basal lamina.

Immunohistochemical localization of ATPase in human dysfunctional corneal endothelium.

Previous light microscopic autoradiographic results from our laboratory have shown a quantitative decrease in ATPase pump site density in dysfunctional corneal endothelium. In order to develop antibody techniques to correlate these findings with electron microscopic localization of pump sites, three types of corneas with dysfunctional endothelium (Fuchs' endothelial dystrophy, aphakic and pseudophakic bullous keratopathy) and two types of corneas with functional endothelium (age matched eye bank and keratoconus) were obtained at the time of transplant surgery. Corneas were fixed, frozen and cryostat sections were incubated in a rabbit kidney ATPase primary antibody followed by incubation in an HRP-labeled secondary antibody. Functional endothelia showed dense antibody labeling along the lateral cell membranes and there was a substantial reduction in labeling along the lateral membranes of dysfunctional endothelia. Positive tissue controls of rabbit kidney showed HRP reaction product in the convoluted tubules. Control tissue, incubated in either non-immune serum or primary antibody preabsorbed with ATPase, confirmed the specificity by having diminished or no reaction product. These results provide immunohistochemical confirmation of our autoradiographic data showing a quantitative reduction in pump site density on dysfunctional corneal endothelium.

ATPase pump site density in human dysfunctional corneal endothelium.

Proper corneal hydration is maintained by a Na, K-ATPase pump located in the lateral membranes of the endothelial cells. In dysfunctional corneas this pumping action appears to break down as the corneas become edematous. In order to provide quantitative and qualitative data on the Na, K-ATPase pump site density on dysfunctional and functional human corneal endothelial cells, the present study has employed both autoradiographic and histochemical techniques. Computer-assisted morphometrics and statistical analysis showed that there was a significant reduction (P less than 0.001) in 3H-ouabain binding, and thus ATPase pump sites, in the three types of corneas (Fuchs' endothelial dystrophy, aphakic and pseudophakic bullous keratopathy) with dysfunctional endothelia as compared to both types of corneas (eye bank, keratoconus) with functional endothelial cells. There were no significant differences amongst the dysfunctional types or between the two functional types of corneal endothelial cells in respect to density of silver grains. Histochemical staining for ATPase showed less p-nitro-phenylphosphatase histochemical reaction product present on dysfunctional endothelial lateral membranes than in the functional cells.

Impact of cataract surgery with lens implantation on vision and physical function in elderly patients.

We conducted a prospective study of 293 elderly patients undergoing cataract surgery with intraocular lens implantation to determine the impact of the surgery on vision and on subjective and objective measures of patient function. Visual acuity in the surgical eye improved from a mean of 20/100 before surgery to 20/40 four months after surgery; improvement was maintained at one year. Positive changes occurred in some subjective measures of patient function, such as reported activities of daily living and patient report of vision-dependent activities, but these changes were modest. The most marked changes in patient function occurred in objective measures of function. Mental status had improved not quite significantly at four months but significantly at one year. Timed manual performance improved dramatically and significantly at four months and one year. Cataract surgery was associated with improved vision and improved objective function in most patients by four months after surgery, and these improvements were maintained at one year.

Effect of intraocular lens convexity on posterior capsule opacification.

A consecutive series of 248 eyes undergoing extracapsular cataract surgery and posterior chamber lens implantation were evaluated. One hundred eight eyes had the implant placed with the convex side posterior and 140 with the convex side anterior. The incidence of capsulotomy required in the convex posterior group was significantly less than in the convex anterior group, 17.6% versus 29.3% (P value less than 0.05). The type of posterior capsule opacification in the two groups was also different. In the convex posterior group, a smooth fibrous type of capsular opacification predominated; in the convex anterior group, epithelial proliferation and pearl formation as well as wrinkling of the capsule frequently occurred. The difference in opacification was reflected in the preoperative visual acuity: convex posterior 20/80, convex anterior less than 20/200 (P less than 0.05).

Freeze-fracture quantitative comparison of rabbit corneal epithelial and endothelial membranes.

The intramembrane structure of epithelial apical, lateral and basal membranes was compared with that of the endothelium in normal rabbit corneas. The cytoplasmic membrane leaflet (P-face) of apical epithelial membrane displays randomly scattered intramembrane particles over the apical microvilli. Apical endothelial P-face membranes, by comparison, are covered by densely-packed particles. The lateral membranes of superficial epithelial cells display delicate tight junctions, punctate particle aggregates characteristic of desmosomes, and small gap junctions. Background intramembrane particles are randomly distributed and not densely packed. Large gap junctions and fewer desmosomes are present on deeper epithelial cells, including basal cells. By comparison, the endothelial lateral membranes display densely-packed, background particles and numerous triangular-shaped, gap junctional particle aggregates bordered by strands of particles resembling tight junctions. At the apical border, long anastomosing tight junctional strands are present. The basal E-face membranes of both cell layers adjacent to the basement membrane are similar in that they have sparse particles and vesicle fusion sites. Small particle aggregates are present on basal epithelial membranes at sites of hemidesmosomes. Quantitation of particle size and density on P-face apical and lateral membranes of each cell layer reveals that particle density on endothelial membranes is more than twice that on epithelial membranes. Particle sizes are similar on both cell layers and mean diameter ranges from 8.7 to 9.2 nm. The differences in intramembrane particle density and junctional aggregates on epithelial and endothelial membranes reflect membrane constituents associated with functional differences of these two cell layers.

Electron microscopy of corneal surface microdiathermy.

Microdiathermy has recently been shown to be effective in the treatment of persistent corneal epithelial recurrent erosion. In order to determine the mechanism of action of microdiathermy on the anterior surface of the cornea, rabbit eyes were treated with microdiathermy and the course of corneal tissue repair studied by electron microscopy. Shortly after treatment, the epithelium is edematous and necrotic and the lamina densa is thickened. Within 24 hours, the epithelium appears healed and some hemidesmosomes are present, but the lamina densa is still thickened. At two weeks the epithelial surface is undulated and protrudes into the stroma in areas where the lamina densa is disrupted. Hemidesmosomes are absent in these regions. Activated fibroblasts are present in superficial stroma. At four weeks following microdiathermy, there is segmental deposition of new lamina densa and a connective tissue zone between the newly deposited lamina densa and the old lamina densa. Hemidesmosomes are present only in areas of newly deposited lamina densa. Between six weeks and three months the epithelial basal surface becomes more uniform with mature hemidesmosomes and the new lamina densa is complete. The old lamina densa remains below it but is no longer present by six months. The mechanism of action for microdiathermy in recurrent erosion is believed to be as follows: in the treated area, epithelium and activated fibroblasts secrete a new connective tissue layer, which provides a suitable substrate to which the epithelium can adhere until it secretes a new lamina densa and hemidesmosome formation can occur.