Improvement of human corneal endothelium in culture after prolonged hypothermic storage.

PURPOSE: To evaluate whether the organ culture method for human cornea preservation may be applied to corneas stored for several days at 4 degrees C. METHODS: The cell density, viability, and morphology of corneal endothelium were examined in 140 human corneas stored at 4 degrees C for the minimal time required for transport to the bank and for the preliminary controls of cornea status (1.6 +/- 1.1 days) and in 46 corneas preserved at 4 degrees C for 6.1 +/- 1.9 days in Optisol-GS. The evaluation was repeated after 19.7 +/- 9.1 days of incubation at 31 degrees C in a culture medium containing 2% newborn calf serum. RESULTS: After the hypothermic storage the corneal endothelium had a mean density of 2475 +/- 159 cells/mm2 without significant difference between the short and the long-term incubation. Several corneas of the two groups showed signs of endothelium degeneration and were positive to trypan blue test. After the incubation at 31 degrees C, the corneas with endothelial degeneration decreased by 52.2% and those positive to trypan blue decreased by 21.7%. Polymorphism (enlarged endothelial cells) increased from 9.6% to 14.5% of the corneas. The remodeling of the endothelium led to a 6.7% decrease in cell density. These results were similar after short-term and long-term storage at 4 degrees C. CONCLUSIONS: Organ culture was effective in improving corneal endothelium when the hypothermic storage was prolonged to the upper temporal limit for this procedure (7-10 days). These results may encourage the possibility of an eye bank to allocate the available cornea pool, thus decreasing the risk of discarding precious material.

Further evaluation of amniotic membrane banking for transplantation in ocular surface diseases.

Objective: To define the best conditions for amniotic membrane preparation, storage and banking in its use for corneal reconstruction. Methods: Amniotic membrane pieces were prepared under sterile conditions from placentas selected on the basis of donor medical and social history, serology, microbiological tests and histology. The pieces were kept at -140 degrees C but before grafting they were thawed and stored at 4 degrees C in RPMI medium, to have a preparation usable within 72 h. This procedure was validated by testing its therapeutic effectiveness in 25 patients 13 of which had corneal ulcers of various origin, 3 had sequelae of herpes simplex keratitis, 3 band keratopathy and 6 corneal stem cell deficiency due to chemical or thermal burns. Results: The preparation showed appreciable anti-inflammatory and analgesic effects. In the absence of corneal stem cell deficiency a stable re-epithelialisation was achieved in 15 out of 19 patients. When the limbus was lesioned, the amniotic membrane decreased vascularization and increased the number of corneal epithelial cells only in 1 of the 6 patients. No adverse reactions attributable to the tissue were recorded. Conclusions: A ready-to-use amniotic membrane preparation stored at 4 degrees C after cryopreservation has been tested in corneal reconstruction. Like the amniotic membrane thawed immediately before grafting, this preparation displayed full therapeutic effect in epithelial defects with stromal ulceration but without severe limbal stem cell deficiency. In two years banking activity 463 pieces of the preparation were successfully distributed to 90 Italian hospitals.

Epidural catheter direction and local anesthetic dose.

BACKGROUND AND OBJECTIVES: The volume of epidural local anesthetic required to achieve a particular dermatomal sensory level varies significantly between patients. Studies have demonstrated random cephalad or caudad migration and direction of the epidural catheter. However, no studies have investigated the relationship between catheter direction and total volume of local anesthetic. METHODS: The study was approved by the Institutional Review Board for the Protection of Human Subjects. After obtaining informed consent, 28 patients (13 men, 15 women) scheduled for elective total knee or hip replacements were enrolled. The epidural catheter was inserted to a depth of 3 cm at L2-3 using a standard 18-gauge Tuohy needle with bevel directed either cephalad or caudad. After test dose, incremental volumes of 0.75% bupivacaine were injected to achieve a sensory anesthetic level of T6, and the total volume of drug was recorded. Immediately after surgery, 1 ml of omnipaque dye was injected through the catheter for x-ray determination of catheter-tip position. Statistical analysis used the Student's t-test and the Wilcoxon's rank sum test. RESULTS: Ages varied from 47 to 79 years (mean age, 66.5 +/- 8.51 years). Weight ranged from 51 to 125 kg (mean weight, 81.7 +/- 14.1 kg). Height ranged from 143 to 188 cm (mean height, 164.2 +/- 10.6 cm). All patients had good surgical anesthesia. Statistically significant differences (p < .001) were seen between average volumes necessary to achieve a T6 sensory level for catheters in the caudad direction (19.2 ml, n = 9) and in the cephalad direction (13.7 ml, n = 19). No statistical correlations could be demonstrated between total dose of local anesthetic and age, sex, weight, or height. CONCLUSIONS: The authors conclude that epidural catheter direction influences the total dose of local anesthetic required to achieve a specific sensory level in epidural anesthesia.