Enrichment of Pb(II) ions using phthalic acid functionalized XAD-16 resin as a sorbent.

A simple and reliable method has been developed using polymeric material containing phthalic acid as a chelating agent to concentrate ultratrace amounts of lead ions in aqueous solutions. After characterization by CHN, IR, and thermal studies, the static and dynamic sorption behavior of Pb(II) ions onto new synthetic resin has been investigated. The sorption has been optimized with respect to pH, shaking speed, and contact time between the two phases. Maximum sorption is achieved from solution of pH 5-8 after 10 min agitation time. The lowest concentration for quantitative recovery is 5.8 ng cm(-3) with a preconcentration factor of approximately 850. The kinetics of sorption follows the first-order rate equation with the rate constant k=0.58+/-0.04 min(-1). The variation of the equilibrium constant K(c) with temperature between 10 and 50 degrees C yields values of DeltaH, 52.4+/-1.65 kJmol(-1), DeltaS, 186+/-5.21 Jmol(-1)K(-1), and DeltaG(303K), -4.15+/-0.002 kJmol(-1). The sorption data of Pb(II) ions in the concentration range from 2.41x10(-6) to 1.44x10(-4) molL(-1) follows the Langmuir, Freundlich, and Dubinin-Radushkevich (D-R) isotherms at all temperatures investigated. The sorption of Pb(II) ions onto synthesized resin in the presence of common anions and cations has also been measured. The possible sorption mechanism of Pb(II) ions onto phthalic acid modified XAD-16 is also discussed. The sorption procedure is utilized to preconcentrate Pb(II) ions prior to their determination in automobile exhaust particulates by atomic absorption spectrometry using direct and standard addition methods.

Vascular endothelial cell growth inhibitor of normal and pathologic human vitreous.

Normal and pathologic human vitreous have been analyzed for the presence of a low-molecular weight inhibitor of aortic endothelial cell proliferation. Vitreous was subjected to gel chromatography and the material appearing in the retarded volume (less than 13,000 daltons) was tested for its ability to inhibit tritiated thymidine incorporation into DNA by calf aortic endothelial cells. Depending on the sample of vitreous analyzed, one or more fractions showing inhibitory activity were identified in each case.

Changes of MK medium during storage of human cornea.

By comparing the composition of McCarey-Kaufman (MK) medium before and after corneal storage we attempted to identify specific physiological changes in the medium as predictors of tissue damage. We also tried to determine if hydrocortisone (a lysosomal membrane stabiliser) added to the medium could reduce tissue damage during storage. Corneas (human and rabbit) were stored in the MK medium with and without hydrocortisone for 4 days at 4 degrees C. The water and nitrogen contents of the stored cornea were compared with those of the fresh cornea. The medium was analysed before and after corneal storage to determine the concentrations of glucose, protein, and amino acids as well as pH and osmolarity. Scanning electron microscopy (SEM) was used to estimate the degree of the corneal endothelial cell damage. The nitrogen contents and dry weights of the steroid treated and untreated stored corneas were similar to those of the fresh unstored cornea. The steroid treated cornea contained a lesser amount of water than the untreated cornea. The cornea stored in medium without steroid took up a greater amount of glucose from the medium than the cornea stored in medium with steroid. As compared with their concentrations in the fresh unused medium the concentrations of leucine, lysine, and glycine were lower and that of glutamic acid was higher in both the media used for corneal storage. However, the steroid treated storage medium as compared with the untreated storage medium had a greater reduction in the lowering of leucine, lysine, and glycine, and a lesser reduction in the increase of glutamic acid. Steroid treated medium also had a lesser amount of protein released from the stored cornea. Changes in the pH and osmolarity of the media before and after corneal storage were not remarkable. SEM showed that the endothelial cells of the cornea stored in the medium containing steroid were less damaged than those of the cornea stored in the medium without steroid.

A morphometric study of endothelial cells of human corneas stored in MK media and warmed at 37 degrees C.

No study has yet been done to investigate the changes in endothelial cell size, perimeter, and density that may result from the warming of corneas in MK (McCarey-Kaufman) medium for specular microscopy. In the present investigation eye bank eyes were stored in MK medium at 4 degrees C and rewarmed daily for six days at 37 degrees C before specular photography of the endothelium was performed. These photographs were compared with wet mount preparations stained with trypan blue and alizarin red made from the same corneas and those stored without rewarming for six days. In addition all corneas were qualitatively analysed with the scanning electron microscope (SEM). The data from serial specular photography were insufficient to allow significant conclusions to be drawn about day to day changes in cell morphology. However, analysis of wet mount preparations revealed that cell density and perimeter varied significantly between those corneas rewarmed daily and those held in cold storage for six days. SEM studies showed an intact cell monolayer with cell loss along the folds of corneal endothelium. We therefore concluded that repeated rewarming at 37 degrees C of corneas stored in MK medium at 4 degrees has a deleterious effect on cell morphology and that folds induced by swelling of corneal tissue result in endothelial cell damage with some loss.

Quantitative analysis of collagen content and amino acids in trabecular meshwork.

The purpose of this study was to compare collagen content in the TM of normal and glaucomatous eyes, and to establish whether collagen levels change with age. Collagen content was measured in 30 normal and 27 age matched glaucoma trabeculectomy specimens by the sirius red dye binding technique, and in 14 normal and 15 age matched glaucoma specimens by amino acid analysis. Both dye binding data and amino acid analysis showed no statistical difference between normal and glaucoma samples. Age had no significant effect on mean optical densities or on the collagen-specific amino acids proline, hydroxyproline, and hydroxylysine. Amino acid variability, however, was statistically different between the two groups. These results indicate that mean collagen levels in the trabecular meshwork of glaucomatous eyes do not differ from those in normal eyes.

Radiochemical separation of strontium by isotopic exchange.

Isotopic exchange has been used for the rapid separation of minute quantities of radio-strontium. A number of variables such as agitation time, concentration of strontium in both phases, mode of preparation and aging of the precipitates, were studied and optimized. With an optimized procedure, exchange yields of 96% and 94% respectively were obtained when strontium sulphate and strontium oxalate precipitates were used for the exchange. Decontamination studies made with radioactive tracers of 15 elements showed that the sulphate precipitate is more selective than the oxalate, most of the elements showing <1 % contamination except (140)Ba-(140)La and (110)Ag which show high contamination. This simple procedure has also been applied for the determination of radioactive strontium in rain-water samples.

The prevention of autolysis in stored corneas by lysosome stabilization. A histochemical study.

We wished to determine if dexamethasome, acting as a lysosome stabilizer, could reduce the release and activation of the lysosomal acid hydrolases, and thus retard autolysis of stored corneas. One cornea (experimental) of a rabbit was soaked in a 2 per cent steroid solution and the other cornea (control) in physiological saline for 3 hours at 23 degrees C. The experimental and control corneas were then processed histochemically to show the localization of the lysosomal marker enzymes beta-glucuronidase and acid phosphatase. Compared to the controls the steroid treated corneas showed reduced enzyme activity suggesting that autolysis during storage had been retarded.

Histochemical localization of hydrolytic enzymes during corneal graft reaction.

We studied hydrolytic enzymes in the graft bed during the corneal graft reaction. Two enzymes, beta-glucuronidase and acid phosphatase, were used as the markers of lysosomal hydrolytic enzymes. These were traced histochemically at various times before and after the onset of the graft reaction in a corneal xenograft model. Results showed a relationship between the degree of graft opacity, cellular infiltration and enzyme localization. It appeared that during the early stages of graft reaction, the granulocytes, and during the late stages, the agranulocytes were the main source of the enzymes.

A corneal model for studying the interaction between the endothelium and sensitized lymphocytes.

We wished to develop a simple model for morphological studies on the interaction between the corneal endothelium and specifically sensitized lymphocytes. Rabbits were transplanted with orthotopic allogeneic skin or corneal grafts. After the onset of the transplantation reaction, lymphoid cells from the recipient's spleen or preauricular lymph nodes were harvested and placed on the endothelial surfaces of the donor and recipient corneas which were incubated in tissue culture medium at 37 degree C for 4 hours before being examined by vital staining or scanning electron microscopy. Compared to the recipient, the donor endothelium contained more lymphoid cell aggregates and more corneal cells were damaged. The in vitro model seemed suitable for studying the cytotoxic behaviour of sensitized lymphocytes confronting the corneal endothelium.

Effects of acidic lake water on the eye.

The normal eyes of 6 men and 21 rabbits were exposed to samples of lake water, one eye to a sample of pH 4.6 and the other to a sample of pH 6.3. The men's eyes were exposed for 5 minutes on four occasions a week apart, whereas the rabbits' eyes were exposed for 15 minutes either on one occasion or once a day for 7 days. In the humans neither sample of water produced symptoms or signs of an adverse effect on the external eye tissues, apart from brief conjunctival congestion after every exposure. In the rabbits the two samples did not appear, in general, to have different effects on the ocular tissues, as judged from the osmolarity and cell count of the tears, conjunctival congestion, corneal staining with fluorescein, corneal permeability and histologic features of the cornea. In a few instances differences were observed, but their pathological significance was not apparent. These data suggest that lake water of a pH as low as 4.6 may not harm healthy eyes, however, larger and broader studies are essential.

Damage to corneal endothelial cells by lysosomal enzymes in stored human eyes.

We wished to see if the concentration of lysosomal enzymes in the aqueous humour had any relation to the number of dead corneal endothelial cells in stored eyes from human donors. Forty pairs of eyes were obtained: one eye of each pair was tested immediately and the other was tested after storage in a moist chamber at 4 degrees C for up to 10 days. The aqueous humour of each eye was aspirated and the concentration of acid phosphatase (a marker of lysosomal hydrolytic enzymes) was measured. Simultaneously the numbers of dead and living cells per unit area of the corneal endothelium were counted following their staining with trypan blue and p-nitroblue tetrazolium respectively. As the storage time increased, the concentration of acid phosphatase in the aqueous humour increased and the number of living corneal endothelial cells decreased. The number of living cells decreased to about 50% at an average enzyme concentration of 9 X 10(-3) muM/h. The eyes stored for less than 3 days were the least damaged.

Quantitative correlation between the reduction of nitroblue tetrazolium and the oxygen uptake of corneal cells.

We wished to measure the amount of reduced nitroblue tetrazolium (NBT) formed in corneal cells and to relate it to the viability of the cells in terms of their oxygen uptake. Two full-thickness corneal discs of identical size (3, 4, 5 or 6 mm in diameter) were cut from the eyes of guinea pigs. One disc was processed for the measurement of its reduced NBT and cellular nitrogen content; the other was processed for the measurement of its oxygen uptake and deoxyribonucleic acid (DNA) content. As the diameter of the corneal disc (that is, the number of cells) increased, the quantity of reduced NBT, nitrogen and DNA and the oxygen uptake increased proportionality. Since each of these measures correlated positively with each other (P less than 0.01) we were able to estimate the number of viable cells in a given cornea by measuring the amount of reduced NBT (formazan granules) formed in the cells.

In-vitro comparative study of a locally prepared corneal storage medium and Optisol.

OBJECTIVE: To compare the in-vitro safety and efficacy of two corneal storage media, Optisol and H-Sol, a chondroitin-sulfate-based medium containing hydrocortisone prepared at the Eye Bank of Canada (Ontario Division). DESIGN: Twenty paired corneas from human donors (mean age 67.9 years) were randomly assigned for storage in corneal viewing chambers at 4 degrees C in Optisol (10 corneas) or H-Sol (10 corneas). The storage media were masked, and all measurements were done in a blinded fashion. OUTCOME MEASURES: Corneal clarity and thickness (measured at 0, 2, 4, 8 and 12 days), endothelial cell density and morphology (analysed at days 0 and 12). At day 12 cell viability was determined by staining with trypan blue and alizarin red S, and three randomly selected corneas from either medium were examined by scanning electron microscopy and transmission electron microscopy. RESULTS: Corneal thickness increased significantly from day 0 to day 12 in both Optisol and H-Sol, and corneal clarity decreased significantly in both media over this period (p < 0.05). At days 2, 4, 8 and 12 the corneas stored in Optisol were significantly thinner than those stored in H-Sol (p < 0.05). There were no significant differences between the media in any of the other indices studied. Endothelial cell density decreased significantly in both Optisol and H-Sol (p < 0.05). There were no within-group differences in percentage of cell loss, coefficient of variation of cell area, figure coefficient or percentage of hexagonal cells. CONCLUSIONS: The difference in corneal thickness between Optisol and H-Sol may have been due to the higher concentration of chondroitin sulfate in the former (2.5%, compared with 2% in H-Sol) or perhaps to the addition of other components to Optisol that are not present in H-Sol. Efforts continue to improve the formulation of H-Sol. Further studies are necessary to assess its safety and efficacy in vivo.

Can steroid reduce endothelial damage in stored corneas? Effect on cell viability and ultrastructure.

We studied the effect of steroid on the viability and integrity of the endothelium of variously stored corneas of different species of animals and man. Paired corneas of rodents and humans were used,--one cornea (experimental) being treated with steroid and the other (control) not. Rat and guinea pig corneas were kept at 37 degrees C for one hour in phosphate buffered saline with or without hydrocortisone 21-sodium succinate (10-6M). Human corneas were kept at 4 degrees C for 4 days in M-K medium with or without hydrocortisone 21-sodium succinate (10-6M). The viability of the endothelia (rat and guinea pig) was tested with para Nitro Blue Tetrazolium and trypan blue dye exclusion tests. The ultrastructural changes in the control and experimental endothelial (guinea pig and human) were examined by scanning electron microscopy. Viable cells P less than 0.001) were more numerous and ultrastructural alteration less in the endothelia of the steroid treated corneas compared to the controls.

Purpurogallin as a cytoprotector of cultured rabbit corneal endothelium.

We examined the protective properties of purpurogallin, a naturally occurring phenol, in delaying necrosis of cultured corneal endothelial cells caused by oxygen free radicals. Endothelial cell cultures were prepared from New Zealand white rabbits using microcarrier cell culture techniques. Corneal endothelial cells were treated with hypoxanthine (2 mM) and xanthine oxidase (67 IU/L) to generate free radicals. The criteria for cell necrosis were cytoplasmic shrinkage, dissolution of plasma membranes and presence of "haloes" around the cells on phase contrast microscopy, confirmed by transmission electron microscopy. More than 95% of second-generation cells exhibited morphologic evidence of necrosis within 4.62 +/- 0.82 minutes after exposure to oxyradicals. The addition of purpurogallin (0.25 or 1.0 mM) significantly increased time to cell necrosis to 8.18 +/- 0.83 and 11.59 +/- 1.71 minutes respectively (p < 0.05). Further studies are under way to determine whether purpurogallin may be useful in preventing endothelial cell damage in corneas preserved for corneal transplantation.

Biochemical analysis of the cornea stored in steroid medium.

We wished to measure changes in the tissue mass and the deoxyribonucleic acid and protein content of guinea pig corneas stored for up to 21 days in tissue culture medium with or without 1 microM hydrocortisone. Full-thickness discs 4 mm in diameter were cut from the corneas for measurement of the three variables. Whether the disc came from a steroid-treated cornea or not, and whether it came from a fresh cornea, hydrocortisone did not appear to have any adverse effect on the features studied.

Does the number of cells in the cornea decrease with age? A biochemical study.

We used biochemical techniques to investigate whether the cornea becomes more acellular with age. Guinea pigs of different body weights (age) were killed. From each cornea, a central full-thickness button (4 mm in diameter) was punched out. The DNA content of the corneal buttons was determined. Using spleen cells, the DNA content per diploid cell of guinea pig was also estimated. It was found that as the body weights of the animals increased, the amount of DNA per corneal disc decreased. This indicated that as the animal grows older the total cellular content of the cornea is reduced.

Toxicity and pharmacokinetics of intravitreally injected ciprofloxacin in rabbit eyes.

To assess the toxicity of intraocular injection of ciprofloxacin, 22 New Zealand white rabbits received midvitreal injections of 100, 200, 400, 800 or 3200 micrograms of ciprofloxacin in 0.1 mL of distilled water (39 eyes) or 0.1 mL of distilled water only (5 eyes). The ocular pharmacokinetics of intravitreally injected ciprofloxacin was determined by aqueous humour and vitreous sampling 1, 2, 4, 8, 12, 18 or 24 hours after midvitreal injection of 100 micrograms of the drug in one eye each of 25 New Zealand white rabbits. The samples were analysed by means of a disc diffusion bioassay. No ocular damage was noted on ophthalmoscopy at any of the concentrations tested. Histologic study showed mild, transient vacuolation of the nerve-fibre layer in all eyes, including the control eyes, 2 hours after injection; at 24 hours no vacuolation was evident except at concentrations of 800 and 3200 micrograms, at which plexiform layer damage was evident. Peak aqueous and vitreous levels of ciprofloxacin were obtained at 1 hour (0.59 and 27.26 micrograms/mL respectively); the vitreous level fell to below 1.0 micrograms/mL 12 hours after injection. We conclude that intravitreally injected ciprofloxacin may be a safe and useful antibiotic in the treatment of aminoglycoside-resistant bacterial endophthalmitis.

Changes in the corneal Na-K ATPase levels in eyes stored in moist chamber at 4 degrees C.

This report deals with a chronological measurement of Na-K ATPase enzyme activity in human and bovine corneas stored in a moist chamber at 4 degrees C. Paired human and bovine eyes were sterilized by the standard eye bank procedure and stored up to 6 days. At the desired time, the corneal endothelium was assayed for Na-K ATPase activity. The protein content of each tissue sample was also determined. In a parallel set of experiments, the viability of identical stored corneas was determined by trypan blue and alizarin red staining technique, and morphometric analysis was done to quantify the extent of the corneal endothelial damage. The human corneas showed that there was a significant progressive decrease in the Na-K ATPase activity as the storage time increased. The decrease was related to morphological endothelial damage.