Examination of murine tear film.

PURPOSE: To define spatially any free aqueous layer in murine tear film. METHOD: A pre-zeroed microelectrode was touched to the superficial corneal epithelium and then raised in steps of 1 micro m through the murine tear film into the air and then retraced along the same path. Other murine tear films were partially probed with a spatial resolution of 0.1 micro m. The reference microelectrode was placed in a fragment of 3% polyacrylamide gel equilibrated against 154 mM NaCl and located on the nasal quadrant of the scleral conjunctiva. Other murine corneas were quick frozen in melting isopentane and freeze substituted or pretreated with cetylpyridinium chloride and then examined by transmission electron microscopy. RESULTS: The recorded electrical profiles of the tear film were reproducible in each preparation and showed a relatively uniform positive electrical potential throughout their whole thickness, except within 0.5 micro m of the epithelial surface when the potential reversed to negative values. The thickness of mouse tear film averaged 7.4 +/- 0.8 micro m (mean +/- SD, n = 40). The electron microscope images showed the murine tear film to have a relatively uniform positive electron density throughout the thickness. CONCLUSIONS: Electrical profiles of the murine tear film presented no evidence of a separate free aqueous phase. The tear film is observed as an aqueous gel that includes anion-exchanging polyelectrolytes throughout most of its thickness, but within 0.5 micro m of the epithelial surface, it changes to cation-exchanging polyelectrolytes. Electron microscope images provide some supporting evidence.

Cryopreservation of cornea: a low cooling rate improves functional survival of endothelium after freezing and thawing.

AIM: To investigate the influence of low cooling rates on endothelial function and morphology of corneas frozen with propane-1,2-diol (PROH). METHODS: Rabbit corneas, mounted on support rings, were exposed to 1.4mol/l (10% v/v) PROH, seeded to initiate freezing, and cooled at 0.2 or 1 degrees C/min to -80 degrees C. Corneas were frozen immersed in liquid or suspended in air. After being held overnight in liquid nitrogen, corneas were warmed at 1 or 20 degrees C/min. After stepwise removal of the cryoprotectant, the ability of the endothelium actively to control corneal hydration was monitored during normothermic perfusion. Morphology was assessed after staining with trypan blue and alizarin red S, and by specular microscopy during perfusion. RESULTS: Functional survival was achieved only after slow cooling (0.2 degrees C/min) with the cornea immersed in the cryoprotectant medium, and rapid warming (20 degrees C/min). These conditions also gave the best morphology after freezing and thawing. CONCLUSION: Cooling rates lower than those typically applied to cornea improved functional survival of the endothelium. This result is in accord with previous observations showing the benefit of low cooling rates for cell monolayers [CryoLetters 17 (1996) 213-218].

Recovery of endothelial function after vitrification of cornea at -110 degrees C.

PURPOSE: To determine whether endothelial function is retained after ice-free cryopreservation of cornea by vitrification at -110 degrees C. METHODS: Rabbit corneas, mounted on support rings, were exposed to a solution containing 6.8 M propane-1,2-diol (PROH) and cooled at approximately 7 degrees C/min to -110 degrees C, which was below the glass transition temperature (T(g)) of the solution. After rewarming at approximately 12 degrees C/min and removal of the PROH, endothelial function was assessed by monitoring corneal thickness during perfusion at 34 degrees C. RESULTS: Addition and removal of 6.8 M PROH without cooling to -110 degrees C did not markedly impair endothelial function, although corneas were thicker than control samples. There was no visible crystallization of ice during cooling to -110 degrees C; but a few small, discrete sites of crystallization remote from the endothelium, were observed during warming. After removal of the PROH, corneas approximately doubled in thickness during the first 3 hours of perfusion, but they then started to thin, which suggested active control of stromal hydration by the endothelium. This was confirmed in a further set of experiments by removal of bicarbonate ions from the perfusate at this point, which resulted in further swelling at +58 +/- 2 microm/hour (SD; n = 4). Restoring bicarbonate to the perfusate halted this swelling, and the corneas then thinned at -13 +/- 2 microm/hour (n = 4). Morphologically, staining with trypan blue and alizarin red S showed an apparently intact endothelial monolayer. CONCLUSIONS: Rabbit corneal endothelium tolerated exposure to 6.8 M PROH, and endothelial function was evident after vitrification at -110 degrees C. Preliminary morphologic results with vitrified human cornea also showed retention of endothelium.