Endothelial Pump Research Articles

Corneal endothelial function after extracapsular cataract extraction and phacoemulsification

Purpose: To compare the morphology and function of the corneal endothelium in the early postoperative period after extracapsular cataract extraction (ECCE) and phacoemulsification.Setting: University Eye Clinic of Trieste, Italy.Methods: In this prospective, randomized study of patients scheduled for cataract surgery, 40 patients were divided into two groups of 20 patients each. Group 1 had ECCE and Group 2, phacoemulsification; both had capsular bag intraocular lens (IOL) implantation. Preoperatively and 7 and 30 days postoperatively, a complete ophthalmological examination, endothelial specular microscopy, ultrasonic pachymetry, and anterior segment fluorophotometry were done. Visual acuity, endothelial cell density, cell size variation coefficient, corneal thickness, endothelial permeability coefficient, and endothelial pump function were studied.Results: Visual acuity was better 7 days after phacoemulsification than after ECCE, but no differences were observed after 30 days. No significant differences in postoperative loss of endothelial cells were found between the two groups. Coefficient of variation in size, corneal thickness, and endothelial permeability significantly increased in both groups 7 days postoperatively, but only in the ECCE group 30 days postoperatively; the differences between the two groups were statistically significant. Endothelial pump function significantly increased after 7 days in only the phacoemulsification group.Conclusions: Functional endothelial failure occurred in the early period after ECCE. Phacoemulsification seemed to minimize postoperative functional damage to the endothelium.

Corneal endothelial protection by different viscoelastics during phacoemulsification

Purpose: To evaluate corneal endothelium morphology and function after phacoemulsification using different viscoelastics.Setting: Eye Clinic, University of Trieste, Italy.Methods: This prospective, randomized study included results of preoperative and postoperative (7, 30, and 90 days) examinations of 66 patients scheduled for phacoemulsification without other ocular pathology; 8 patients were excluded. Patients were randomly assigned to one of four groups based on type of viscoelastic used: 1% sodium hyaluronate (Healon®); 1.4% sodium hyaluronate (Healon GV®); 4% sodium chondroitin sulfate-3% sodium hyaluronate (Viscoat®); 2% hydroxypropyl methylcellulose (Hymecel®). Mean cell density and cell size variation coefficient were determined by specular microscopy; central corneal thickness, by ultrasonic pachymetry; and endothelial permeability coefficient and active pump function, by anterior segment fluorophotometry.Results: There were no significant differences in postoperative mean cell loss among the groups. The cell size variation coefficient was altered in all groups 7 days after surgery and was still impaired at 30 days in the Hymecel group. A significant increase in mean corneal thickness, endothelial permeability, and active pump function occurred in the Healon and Hymecel groups 7 days after surgery. These parameters were still altered 30 days after surgery in the Hymecel group. Endothelial functional alterations did not occur in the Healon GV or Viscoat group.Conclusion: Viscoat and Healon GV are effective in minimizing functional damage of endothelial structure in the early and medium-term postoperative period.

Effect of baroreceptor activation and systemic hypotension on plasma endothelin 1 and neuropeptide Y.

To determine whether endothelin (ET-1) and neuropeptide Y (NPY) release are controlled by the carotid sinus (CS) baroreceptor or local endothelial mechanisms, we isolated and pump perfused the CS in eight chloralose-anesthetized dogs and controlled systemic arterial pressure (SAP) with an elevated reservoir connected to both femoral arteries. This allowed the SAP to be kept constant while CS pressure was varied from 55.8 +/- 2.0 (low CS) to 192 +/- 1.9 (high CS) mmHg (1 mmHg = 133.3 Pa) or CS pressure to be kept constant while SAP was lowered to 53.9 +/- 1.8 mmHg (low SAP). There was no significant change in ET-1 when CS pressure was varied (control, 2.08 +/- 0.50; low CS, 2.18 +/- 0.51; high CS, 2.11 +/- 0.38 pg/mL), but ET-1 was significantly higher during low SAP (2.93 +/- 0.49 pg/mL, p < 0.05). This increase was not observed with vagi and CS intact in six dogs or with vagi intact and CS constant in four dogs. In contrast, plasma NPY was significantly higher in the low CS condition (619.13 +/- 66.87 pg/mL) versus high CS condition (528.88 +/- 45.19 pg/mL, p < 0.05) and did not change during hypotension. In conclusion, NPY, but not ET-1, is affected by CS baroreceptor manipulation, and plasma ET-1 increases in response to hemorrhagic hypotension when modulating reflexes are abolished.

Evidence for a direct effect of bicarbonate on the rabbit corneal stroma.

Currently prevailing opinion on the mechanism of corneal deturgescence (stromal thinning) is that it is a process mediated by an endothelium-sited fluid pump. This pump is considered to be bicarbonate-dependent and balanced by the passive tendency of the stroma to absorb fluid that leaks from the aqueous across the endothelium. A case is presented that the utility of older key experiments as supportive evidence for this bicarbonate pump is limited by methodological errors in the formulation and use of bicarbonate-containing solutions. Recent studies reveal the pump to be generally unaffected by exogenous bicarbonate levels. Several recent studies confirm that the deturgescence of stroma-endothelium preparations is remarkably sensitive to exogenous bicarbonate and further indicate that the actual hydration of the stroma is inversely related to exogenous bicarbonate levels. These studies also show that it is possible to observe deturgescence in stroma-endothelial preparations which have no apparent net fluid pump activity. Conversely, net fluid pump activity has been measured in preparations which nonetheless fail to show deturgescence. Therefore, there does not appear to be an obligate coupling between deturgescence and endothelial fluid pump activity. The possible role of the endothelium in regulating the bicarbonate movement into the collagen-keratocyte matrix of the corneal stroma (rather than pumping it out) is discussed as a possible interpretation of more recent data.

Effects of Intraocular Irrigants on the Preserved Human Corneal Endothelium

Either a simple balanced salt solution (BSS) or a bicarbonate-buffered, glutathione-containing commercial irrigant (BSS Plus, Alcon Laboratories, Fort Worth, Texas, U.S.A.) may be used during the vitrectomy portion of a corneal transplant procedure. To simulate the conditions present in the anterior chamber during the first few hours after vitrectomy and grafting, we performed in vitro perfusions of stored human corneas using each irrigant and measured corneal thickness over a 3-hour period. Irrespective of the preservation medium used (McCarey Kaufman, Chondroin Sulfate or Dexsol, all from Chiron Ophthalmics, Irvine, California) or duration of storage (2 or 4 days), corneas irrigated with BSS Plus exhibited significantly (p less than .05) decreased thickness compared with their paired mates irrigated with BSS. In ultrastructural studies performed on postperfusion corneas, there was a tendency toward improved surface morphology in the in vitro BSS Plus-perfused tissue. This study shows that in vitro corneal thickness after preservation is significantly irrigant dependent, with BSS Plus providing the essential ingredients to promote the corneal endothelial pump function.

The potential of an equimolar combination of propane-1,2-diol and glycerol as a vitrification solution for corneas

Corneas must first be equilibrated with multimolar concentrations of cryoprotectants if the formation of ice during cryopreservation is to be avoided by vitrification at practicable cooling rates. Rabbit corneas were exposed to equimolar mixtures of the cryoprotectants propane-1,2-diol and glycerol in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mmol/liter sodium bicarbonate, and 6% w v bovine serum albumin. Endothelial function was assessed by monitoring its ability to control stromal hydration during perfusion of the endothelial surface at 34 °C for 6 h. Endothelial morphology was observed by specular microscopy during perfusion and by scanning electron microscopy after perfusion. Endothelial pump activity and structural integrity of the endothelial layer were demonstrated after 20 min exposure at 4 °C to a total concentration of 1.4 mol/liter cryoprotectant (i.e., 0.7 mol/liter propane-1,2-diol + 0.7 mol/liter glycerol). Exposure to 2.0 and 3.4 mol/liter cryoprotectant for 20 min at 4 ° and − 5 °C, respectively, resulted in initial endothelial damage, but this repaired and a functioning endothelial pump was subsequently demonstrated. Although exposure to 4.1 mol/ liter cryoprotectant for 10 min at − 10 °C caused irreparable damage to 2 4 corneas, reduced dilution temperatures together with increased dilution time allowed exposure to 4.8 and 5.5 mol/liter cryoprotectant with retention of endothelial pump activity. Exposure to 6.1 mol/liter cryoprotectant for 10 min at − 15 °C caused endothelial damage which was not mitigated by the presence of 2.5% w v chondroitin sulfate. Endothelial function may be improved by further modification of addition and dilution protocols or by exposure to the cryoprotectants at lower temperatures.

Effect of humidity on the deswelling function of the human cornea

The present study determined the effect of the full range of humidities on the deswelling function of the human cornea. The closed-eye deswelling function and the open-eye deswelling responses for five different levels of humidity (0%, 25%, 60%, 85%, and 100%) were assessed for 8 normal, young-adult subjects. Open-eye corneal deswelling for the 8 subjects was unaffected by relative humidities from 0 to 100%. Therefore, osmotically-driven corneal thinning effect of tear evaporation does not significantly contribute to the deswelling function of the human cornea. We conclude, contrary to recent reports, that the endothelial pump is the primary mechanism that maintains normal corneal thickness and provides recovery from stromal edema.

The cell barrier effect in rabbit corneas kept under eye bank conditions.

The influence of endothelial cell pumping and of cell barrier functions on cultivated corneas has been studied by comparing the rate of weight increase in freeze-treated, ouabain-treated and fresh corneas. Inhibition of the endothelial cell pump activity increased the swelling rate from 0.5% of start weight per h (fresh corneas) to about 2% per h (ouabain-treated corneas), as measured after 24 h in culture medium. The cell barrier effect was estimated by comparing the swelling rate of ouabain-treated corneas after 24 h, and the swelling rate of freeze-treated corneas with the same swelling pressure. The values are 2 and 9.5% per h, respectively. The swelling rate of freeze-treated corneas with different button diameters was found to increase with decreasing corneal button diameter.

Physiological state of the rabbit cornea following 4°C moist chamber storage

Eyes from female albino rabbits (1.9-2.3 kg) were enucleated immediately following euthanasia along with the lids and conjunctiva. The globes were moist chamber-stored (in a 4 degrees C refrigerator with the lids closed and the cornea facing downwards) for 12-500 hr. Central corneal thickness (by ultrasound) increased from 350 to approximately 650 microns within 72 hr but changed little thereafter. In the latter period, the relative fluid pressure of the globe (pneumatonography) dropped to less than 10 mmHg, aqueous bicarbonate (tCO2) levels fell to less than 5 mM and anterior chamber fluid tonicity decreased progressively (especially after 24 hr storage) to reach values of around 200 mosmol l-1 by 7 days. Storma-endothelium preparations of the corneas, after 2 hr of in vitro equilibration with a bicarbonate-Ringer solution (supplemented with glucose, adenosine and glutathione) were evaluated for their ability to undergo deturgescence under silicone oil or to pump fluid against a hydrostatic pressure of 20 cmH2O. Corneal preparations from up to 7 days storage showed rapid (60 to 135 microns hr-1) and complete deturgescence (net change in thickness of 140-180 microns) that was maintained. Thereafter, the ability to show deturgescence declined to zero by 10 days. In marked contrast, endothelial fluid pump activity (of approximately 5 microliters hr-1) was manifest for only 36 hr after which time this function rapidly declined. Most corneas stored for periods longer than 48 hr exhibited a continuous leak (of -1 to -5 microliters hr-1). The results indicate that corneal deturgescence and endothelial fluid pump function are not necessarily coupled in vitro.

Keratopathy of the rabbit cornea following complete eyelid closure

The corneal response to a complete tarsorrhaphy was studied in 46 rabbits for time periods up to 21 days. During the first 7 days of complete eyelid closure corneal thickness increased up to 18.4%, glycogen decreased 32.2%, and lactate rose 27.2%. A steep increase in corneal thickness to 89.8% appeared after 14 days, followed by decreasing values at 21 days, which was concomitant with the formation of extensive vascularized pannus. These results confirm previous findings that the partial pressure of O2 under closed eye conditions is substantially below physiologic requirements, affects endothelial pump capability, and results in major corneal swelling. When silicone contact lenses were fitted immediately before eyelid closure, corneal swelling 2 days after lid closure did not differ from that in eyes without lenses.

Endothelial pump function in rabbit corneas kept under eye bank conditions

Endothelial pump activity of rabbit corneas kept under eye bank conditions was examined. Different swelling rates for medium with and without ouabain were used for the calculation of endothelial pump rate. After an initial weight increase during 24 h, the control corneas kept approximately constant weight for 1 week. The ouabain-treated corneas increased continually, and during the time interval 24-48 h the increase was 41% of start weight. This corresponds to a pump activity of 2.3% of the weight of the normal corneal stroma per h, or to 9 microns of the corneal thickness per h. Each endothelial cell pumped fluid corresponding to 200-300% of the cell volume per h between day 1 (24 h) and day 8. The pump activity was gradually reduced after 8 days. The average weight was the same in both groups after 7 weeks.

Endothelial Function and Aqueous Humor Flow Rate in Patients With Fuchs’ Dystrophy

Using a two-dimensional scanning fluorophotometer, we studied 50 subjects with symmetric ocular involvement of Fuchs' dystrophy without epithelial edema. Twenty-six subjects with confluent or nearly confluent cornea gutata with increased corneal thickness and 24 subjects with mild to moderate cornea guttata with normal corneal thickness were compared to normal control subjects. There were no statistically significant differences in endothelial permeability between the three groups. Corneal thickness was significantly increased in the subjects with confluent to nearly confluent guttae, however. These results suggest that endothelial pump function may be affected in subjects with advanced cornea guttata with stromal edema.

Variations in human corneal endothelial cell morphology and permeability to fluorescein with age

Fluorophotometry with topically applied fluorescein and endothelial cell photography were performed on 80 normal subjects (age 5–79 yr). Variations in endothelial cell morphology and function, flow of aqueous humor, and intraocular pressure were recorded. The mean endothelial cell size was 332·3 ± 46·3 μm 2. A 28% increase in endothelial cell size was measured over the eight decades ( r = 0·53, P < 0·001). The coefficient of variation of cell size also increased with age ( r = 0·41, P < 0·001). The percentage of hexagonal endothelial cells decreased by 14% ( r = −0·48, P < 0·001), while the percentage of pentagonal and heptagonal cells increased by 50% ( r = 0·44, P < 0·001) and 40% ( r = 0·33, P < 0·002), respectively, with age. The mean endothelial permeability to fluorescein was 4·03 ± 0·63 × 10 −4 cm min −1. A 23% increase in endothelial permeability with age was observed ( r = 0·44, P < 0·001). No change in central corneal thickness or endothelial pump rate was found. Flow of aqueous humor remained stable with age, despite a 25% increase in intraocular pressure ( r = 0·50, P < 0·001). Polarization of fluorescence of fluorescein in the corneal stroma decreased with age ( r = −0·46, P < 0·001). We conclude that with age the human corneal endothelium becomes morphologically less regular and may become more permeable to fluorescein.

Sided effects of bicarbonate on rabbit corneal endothelium in vitro

Stroma-endothelium preparations from albino rabbits were studied either in a specular microscope (and measurements made of corneal thinning under silicone oil) or mounted between two half-chambers (to permit direct measurement of endothelial fluid pump activity). Preparations were first equilibrated with bicarbonate-Ringer solutions (supplemented with glucose, adenosine and glutathione and equilibrated with 5% CO2-air). If the preparations were equilibrated with a low (2 mM) level of bicarbonate on the stromal side and variable levels (2 to 50 mM) of bicarbonate on the endothelial side, corneal thinning increased with bicarbonate concentration while fluid pump was found to be constant from 2 to 35 mM bicarbonate. In contrast, if the preparations were equilibrated with a low (2 mM) bicarbonate level on the endothelial side and 2 to 50 mM bicarbonate on the stromal aspect, corneal thinning did not occur but fluid pump declined gradually as a function of stromal bicarbonate levels. These results contrast sharply with the significant corneal thinning and fluid pump that can be measured if both sides of the preparations are equilibrated with 35 mM bicarbonate. These results further emphasise that bicarbonate ions do not simply serve to drive an endothelium-sited fluid pump in the mammalian cornea.

Physiological changes in the cornea of the ageing eye.

Donor human corneas were studied in vitro for the viability of their endothelia. Endothelial ionic permeability (measured by electrical conductivity) and bicarbonate pump activity (measured by short circuit current) were recorded. Paired corneas were used to monitor post-mortem deterioration times and drug therapy immediately prior to death noted, when possible, to identify any possible effect on tissue performance. Donor age was correlated with some loss of endothelial pump capability. In adults it seems that pump capability deteriorates with a half time decay of 72 years (correlation coefficient, 0.69). Typically, between 60 and 90 years, endothelial pump capability may drop from 32 mu amps.cm-2 to 22 mu amps.cm-2, although at any particular age, the natural variation is +/- 6 mu amps.cm-2. Over the same period, endothelial permeability does not deteriorate and there is a suggestion that the endothelial cells may even tighten up with ageing, thereby partially compensating for the suggested ageing diminution in metabolic pump capability. There is no obvious age threshold for endothelial deterioration. The use of paired corneas revealed a 50 per cent loss in endothelial barrier function per 4.8 days of storage, and less than 5 per cent loss of pump capability over a similar period. The eyes were stored whole in a moist chamber at 0-4 degrees C. A significant group of donor eyes had a markedly reduced pump capability which was not age related. The patients had had terminal drug treatments which included digoxin and furosemide.

Effect of inflammation on the corneal endothelial pump and barrier.

Corneal thickness is a reflection of endothelial barrier and pump functions. The corneal edema that occurs during intraocular inflammation is a consequence of the breakdown of one or both of these parameters. Results of this study demonstrate that, during intraocular inflammation induced by an intravitreal injection of bovine serum albumin (BSA), the permeability of rabbit corneal endothelia to inulin was increased. By comparison, treatment with oral aspirin and/or subconjunctival triamcinolone acetonide prevented the endothelial barrier breakdown induced by the BSA. Concomitant with the loss of the barrier function, endothelial ouabain binding decreased in the BSA injected eye, indicating a reduction in endothelial Na/K ATPase pump site density. A subconjunctival injection of triamcinolone prevented this decrease in pump sites. The increase in endothelial permeability and the decrease in pump site density correlated with an increase in corneal thickness. It can be concluded that the intraocular inflammation induced by BSA effects corneal edema by both an increase in endothelial permeability and a decrease in Na/K ATPase pump site density. Subconjunctival triamcinolone is effective in preventing this response.

Corneal epithelial Cl-dependent pump quantified

Pairs of rabbit corneas were perfused and corneal thickness monitored. The epithelial side of the preparation, and after stabilization also the endothelial side, was covered with silicone oil. A constant thinning of the cornea was observed at a rate of 11.1 microns hr-1 (= 1.0 micron L h-1 cm-2) S.D. 2.1, n = 9. This thinning was also present after endothelial removal: 12.3 microns hr-1, S.D. 1.6 (n = 5). Epithelial abrasion virtually abolished the thinning (2.5 microns hr-1, S.D. 1.89, n = 4), as did cooling from 34 to 0 degrees C. When the preparation was perfused with a Cl-free solution (SO4(2-) instead of Cl-, corrected for osmolarity with sucrose) no significant thinning of the preparation was observed (2.4 microns hr-1, S.D. 2.49, n = 4) after covering both surfaces with silicone oil. This simple set of experiments quantified the epithelial pump mechanism. The epithelial pump rate of about 1.2 microns L hr-1 cm-2 has to be taken into account when endothelial pump rates are measured in the in vitro preparation with intact epithelium.

A TEST BED FOR OCULAR DRUGS*

Corneal endothelium plays a vital role in maintaining corneal transparency. We describe here a new in vitro technique for detecting acute effects of ocular drugs on corneal endothelium from animal species and post-mortem human tissue. The technique is based upon trans-endothelial electrical activity and leads as a consequence, to rapid, unambiguous, quantitative assessments of pharmacological effects upon endothelial barrier function and fluid pump activity.

Effect of HEPES buffer on corneal storage in MK medium.

Rabbit corneas were stored for 7 days in either MK medium containing gentamicin or modified MK medium containing HEPES buffer, gentamicin and phenol red. Corneas stored for 7 days in modified MK medium were thicker than corneas stored in MK medium. Corneal endothelial permeability to inulin and dextran was similar following 7 days of storage in either solution. Transmission electron microscopy of corneal endothelial cells stored in either solution showed intact cell membranes and organelles. In vitro perfusion of rabbit corneas in the specular microscope with Krebs Ringer bicarbonate containing HEPES buffer swelled at 17 +/- 1 micron/h, whereas those perfused with Krebs Ringer bicarbonate alone swelled at 7 +/- micron/h. Perfusion with Krebs Ringer bicarbonate containing phenol red did not result in an increased corneal swelling rate. The work indicates that HEPES buffer has an adverse effect on corneal endothelial pumping function, and this results in corneal swelling during storage as well as during perfusion in the specular microscope. The adverse effect appears to be, at least in part, transient: however, the ultimate, long term effect of HEPES buffer on corneas stored prior to penetrating keratoplasty is not known and deserves continued investigation.

Decreased Endothelial Permeability in Transplanted Corneas

We measured the endothelial permeability to fluorescein of 30 clear corneal transplants from one to six years after keratoplasty. The mean permeability of 1.51 +/- 0.82 X 10(-4) cm/min was significantly lower than that of normal corneas (P less than .00001). We detected a statistically significant negative correlation between permeability and mean endothelial cell size (P less than .03), indicating that corneas with larger endothelial cells tended to be less permeable to fluorescein. We calculated a relative endothelial pump rate for each cornea; the pump rate was also decreased and negatively correlated with mean endothelial cell size (P less than .0004).