Abstract
Purpose :
Aqueous outflow facility (C) has been measured in live mice and also enucleated mouse eyes. In our hands C in live mice or enucleated eyes within 120 min of euthanasia is similar when perfusing via the anterior chamber (AC). But we have not studied effects of elapsed time following enucleation. Here we measured C in enucleated mouse eyes following various storage times. For comparison, we also measured C in eyes in live animals.
Methods :
Eyes were enucleated (C57BL/6J mice, ♂, 9-10 months) immediately following euthanasia, and then placed in PBS and stored at 4○C for 1-168h. C was measured using (1) constant flow infusion (CFI), and (2) constant pressure infusion (CPI). For CFI, the AC was cannulated (32G steel needle). Eyes were immersed in PBS to prevent ocular surface evaporation, and infused with 1×PBS from a pump at the following flow rates (F) (nL/min): 200, 240, 280, 320, 360, and 400, until pressure (P) plateaued. The reciprical of the slope of P vs. F returned C. Eyes were then subjected to CPI. An adjustable height manometer with an open PBS reservoir was switched into the infusion line. P was set to 20 mmHg (designated as P1). Tubing under the reservoir was arranged next to a scale. The meniscus level in the tubing was recorded. After 4 min, the level by which the meniscus had dropped (D), and the new pressure (P2) was recorded. Mean P over this period was calculated as (P1+P2)/2. This process was repeated at: 17, 14, 11, and 8 mmHg. F over each 4 min interval (and each value of D) was calculated from π×r2×D (where r = tubing internal radius). The slope of F vs. P returned C. Live animals were perfused similarly, except that (1) animals were anesthetized (ketamine/xylazine (100mg/kg:10mg/kg)), and (2) for CFI, the following flow rates were utilized (nL/min): 40, 80, 120, 160, and 200. Animals were maintained at 37°C. A drop of PBS was placed on each eye to prevent corneal surface evaporation.
Results :
C (nL/min/mmHg) measured by CFI: 18.9±1.5 (Live), 17.8±1.5 (1h PE), 11.2±0.5*** (24h PE), 8.4±0.4**** (72h PE), and 5.9±0.5**** (168h PE)
C measured by CPI: 19.7±2.0 (Live), 18.2±1.5 (1h PE), 12.1±0.5*** (24h PE), 8.8±0.3**** (72h PE), and 6.5±0.5**** (168h PE).
***p<0.001; ****p<0.0001; difference from live eyes (n=8, all groups). PE: Post-Enucleation
Conclusions :
C diminishes as storage time increases in enucleated murine eyes.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.