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Purpose: When keratorefractive surgery is used to treat a central corneal diameter smaller than the resting pupil, visual symptoms of polyopia, ghosting, blur, haloes, and glare can be experienced. Progress has been made to enlarge the area of surgical treatment to extend beyond the photopic pupil; however, geometric limitations can pose restrictions to extend the treatment beyond the mesopic pupil diameter and can lead to impediments in night vision. The size of the treated area that has achieved good optical performance has been defined as the functional optical zone (FOZ). In this study the authors developed three objective methods to measure the FOZ., Methods: Corneal topography examination results from 1 eye of 34 unoperated normal eyes and 32 myopic eyes corrected by laser in situ keratomileusis (LASIK) were evaluated in three ways. First, a uniform axial power method (FOZ(A)) assessed the area of the postoperative cornea that was within a +/-0.5-D window centered on the mathematical mode. Second, FOZ was determined based on the corneal wavefront true RMS error as a function of the simulated pupil size (FOZ(R)). Third, FOZ was determined from the radial MTF, established at the retinal plane as a function of pupil size (FOZ(M))., Results: Means for each of the FOZ methods (FOZ(A), FOZ(R), and FOZ(M)) were 7.6, 9.1, and 7.7 mm, respectively, for normal eyes. For LASIK-corrected eyes, these means were 6.0, 6.9, and 6.0 mm. Overall, an average decrease of 1.8 mm in the functional optical zone was found after the LASIK procedure. Correlations between the FOZ methods after LASIK showed acceptable and statistically significant values (R = 0.71, 0.70, and 0.61; P < 0.01)., Conclusions: These methods will be useful to more fully characterize corneal treatment profiles after keratorefractive surgery. Because of its ease of implementation, direct spatial correspondence to corneal topography, and good correlation to the other more computationally intensive methods, the semiempiric uniform axial power method (FOZ(A)) appears to be most practical in use. The ability to measure the size of the FOZ should permit further evolution of keratorefractive surgical lasers and their algorithms to reduce the night vision impediments that can arise from functional optical zones that do not encompass the entire mesopic pupil.

Purpose: It is assumed that wavefront error data arising from aberrometry are adequately described by a Zernike polynomial function, although this assumption has not been extensively tested. Inaccuracies in wavefront error may compromise clinical testing and refractive correction procedures. The current retrospective study correlates visual acuity with corneal wavefront error and with the residual surface elevation error after fitting with the Zernike method., Methods: Corneal topography maps were obtained from 32 keratoconus cases, 27 postoperative penetrating keratoplasty cases, and 29 postoperative conductive keratoplasty cases (88 total). The best spectacle-corrected visual acuity (BSCVA) for each case ranged from -0.2 to 1.3 logarithm of the minimum angle of resolution (logMAR) units (20/12.5-20/400). Topography was analyzed to determine wavefront error and the elevation fit error for a 4-mm optical zone. The 4th and 10th expansion series were analyzed with the 0th-order (piston) and 1st order (tip and tilt) removed. Linear regression analysis was performed. The difference in root mean square (RMS) error between the 4th- and 10th-order analyses was assessed for both wavefront and elevation fit error., Results: The correlation of BSCVA to wavefront error for 4th-order terms was moderately strong and significant (R2=0.581; P<0.001). The 10th-order correlation for wavefront error had a similar result (R2=0.565; P<0.001), but the regression was not significantly different from the 4th-order result. The correlation of BSCVA to the elevation fit error was strong and significant for the 4th order (R2=0.658; P<0.001). The 10th-order data had a similar result (R2=0.509; P<0.001), and there was no significant difference between the two regressions. Only 72% of the cases showed a shift toward increased wavefront error with the 10th-order series, whereas 18% lost wavefront error. All cases showed a shift toward improved elevation fit with the 10th-order expansion., Conclusions: The wavefront error correlation to acuity was moderately strong, but the corneal elevation fit error also strongly correlated with visual acuity, indicating that Zernike polynomials do not fully characterize the surface shape features that influence vision and that exist in postsurgical or pathologic eyes. In addition, the change in wavefront error when using a larger expansion series was found to increase or diminish somewhat unpredictably. The authors conclude that Zernike polynomials fail to model all the information that influences visual acuity, which may confound clinical diagnosis and treatment.

Purpose: To determine the specific biochemical pathways involved in the initial-phase inflammatory response that causes stromal edema after epithelial debridement of the rabbit cornea., Methods: Adult New Zealand White rabbit corneas were treated with 2 mM synthetic inhibitor of metalloproteinase (SIMP)-1, 1 mM DFU (a specific cyclooxygenase [COX]-2 inhibitor) in 50/50 dimethyl sulfoxide (DMSO)/Ringer's solution, 300 KIU aprotinin (a serine protease inhibitor), 0.05% or 0.10% IL-1 receptor type II solution, 1 mM gliotoxin (a Ras farnesyltransferase inhibitor), or vehicle alone (the control). These were applied topically in vivo in five doses over a 3-hour period except IL-1 receptor type II, which was applied in vitro. After rabbits were killed, the corneas were mounted in perfusion chambers with the endothelium bathed in a modified Ringer's solution and the epithelium bathed with silicone oil. Corneal thickness was measured with an automatic specular microscope. The corneal thickness typically stabilized 1 hour after mounting. After stabilization, the corneal epithelium was removed with a rotating bristle brush and stromal thickness monitored for 1 hour. Paired control corneas were treated similarly. RESULTS. Stromal swelling after epithelial debridement was significantly less in most treated corneas, compared with untreated controls: 18.4 +/- 5.3 microm vs. 28.6 +/- 7.7 microm (n = 6, P = 0.004); SIMP-1, 18.7 +/- 10.2 microm vs. 34.3 +/- 10.2 microm (n = 7, P = 0.02); DFU, 19.3 +/- 10.2 microm vs. 23.5 +/- 8.4 microm (n = 6, P = 0.01); and IL-1 receptor type II (0.05%), 26.2 +/- 5.6 microm vs. 30.4 +/- 5.6 microm (n = 5, P = 0.03) and (0.10%), 26.6 +/- 5.6 microm vs. 32.1 +/- 7.4 microm (n = 8, P = 0.03). Gliotoxin was not effective (21.5 +/- 8.0 microm vs. 21.9 +/- 6.2 microm; n = 5, P = 0.94)., Conclusions: The reduction of stromal edema after topical administration of the inhibitors demonstrates the involvement of the COX-2 enzyme, the matrix metalloproteinase family, plasminogens, and the IL-1 system in the trauma-induced inflammatory response of the rabbit cornea. The inflammatory process in the cornea associated with trauma can proceed along multiple redundant parallel pathways.

Purpose: To investigate the nature and cause of an acute, anomalous stromal edema after epithelial debridement in the rabbit cornea., Methods: Series I: Adult New Zealand White rabbit corneas were mounted in perfusion chambers. The endothelium was bathed with Ringer's fluid, and the outer surface was covered with silicone oil. The epithelium of one eye was débrided with a scalpel before mounting, and the cornea of the fellow eye was débrided with a rotating brush after stabilization in the perfusion chamber. Using specular microscope tracking software, it was possible to measure total swelling and local swelling within the cornea. Series II: Diclofenac sodium ophthalmic solution 0.1% or a placebo was applied topically, 1 drop per 45 minutes for 3 hours before animals were euthanatized., Results: Series I: Corneas with their epithelium scraped with a scalpel before mounting were 37.5 +/- 17.5 microm (n = 6; P < 0.001) thicker in vitro than the stromas of perfused, intact fellow corneas. Epithelial débridement with a rotating brush after mounting resulted in an immediate (within 8 minutes) stromal swelling that plateaued in 1 hour at 31.0 +/- 5.3 microm (n = 6; P < 0.001). Curiously, in six of six corneas, the anterior stroma swelled more than the posterior stroma. In four of six corneas, the posterior stroma thinned. Analysis showed this pattern to be consistent with a sudden increase in anterior swelling pressure or osmotic pressure and to be inconsistent with a change in endothelial transport properties. Series II: Placebo-treated corneas swelled 30.6 +/- 7.7 microm (n = 5) 1 hour after débridement, whereas corneas pretreated with diclofenac sodium swelled only 19.2 +/- 3.1 microm (n = 6; P < 0.008)., Conclusions: The anterior stromal swelling occurs rapidly and near the site of epithelial injury suggesting messenger and/or enzymatic involvement with an effect parallel to apoptosis. Reduction of the swelling response with nonsteroidal anti-inflammatory drugs (NSAIDs) implicates the cyclooxygenase pathway. The swelling is similar to the unexplained acute edema that occurs during inflammation in the rat paw edema model, and may represent a general mechanism for mobilization of inflammatory cells.

Purpose: To investigate whether corneal wavefront aberrations vary with aging., Methods: One hundred two eyes of 102 normal subjects were evaluated with videokeratography. The data were decomposed using Taylor and Zernike polynomials to calculate the monochromatic aberrations of the cornea for both small (3-mm) and large (7-mm) pupils., Results: For a 3-mm pupil, the amount of total aberrations (Spearman rank correlation coefficient r(s) = 0.145; P = 0.103) and spherical-like aberrations (r(s) = -0.068; P = 0.448) did not change with aging, whereas comalike aberrations exhibited a weak but statistically significant correlation with age (r(s) = 0.256; P = 0.004). For a 7-mm pupil, total aberrations (r(s) = 0.552; P < 0.001) and comalike aberrations (r(s) = 0.561; P < 0.001) significantly increased with aging, but spherical-like aberrations showed no age-related changes (r(s) = 0.124; P = 0.166). Simulated pupillary dilation from 3 mm to 7 mm caused a 38.0+/-28.5-fold increase in the total aberrations, and the extent of increases significantly correlated with age (r(s) = 0.354; P < 0.001). Pupillary dilation influenced the comalike aberrations more in the older subjects than in the younger subjects (r(s) = 0.243; P = 0.006), but such age dependence was not found for spherical-like aberrations (r(s) = 0.141; P = 0.115)., Conclusions: Comalike aberrations of the cornea correlate with age, implying that the corneas become less symmetrical along with aging. Spherical-like aberrations do not vary significantly with aging. Pupillary dilation markedly increases wavefront aberrations, and those effects are more prominent in older subjects than in younger subjects.

Purpose: Four videokeratographic methods for keratoconus detection were compared with a neural network approach., Methods: A classification neural network for keratoconus screening was designed to detect the presence of keratoconus (KC) or keratoconus suspects (KCS); a separate cone severity network graded the severity of conelike topography patterns consistent with KC or KCS. Three hundred TMS-1 examinations (Tomey) were randomly divided into training and test sets. Ten topographic indexes were network inputs. Nine categories were used: normal, astigmatism, KC, KCS, contact lens-induced warpage, pellucid marginal degeneration, photorefractive keratectomy, radial keratotomy, and penetrating keratoplasty. KC was subdivided into KC1 (mild), KC2 (moderate), and KC3 (advanced). There were three outputs for the classification network (KC, KCS, and OTHER); target output values of 0 = OTHER, 0.25 = KCS, 0.5 = KC1, 0.75 = KC2, and 1.0 = KC3 were used for the severity network., Results: The best-trained classification network had 100% accuracy, specificity, and sensitivity for the test set. The severity network had mean outputs (+/-standard deviation) of OTHER = 0.02+/-0.02, KCS = 0.21+/-0.05, KC1 = 0.52+/-0.17, KC2 = 0.74+/-0.12, and KC3 = 0.91+/-0.15. The severity network output for all categories was well correlated to the keratoconus prediction index (R = 0.892, P < 0.0001). The classification network had an overall accuracy and specificity significantly better (P < or = 0.005) than the Klyce/Maeda keratoconus index (KCI) test, the Rabinowitz test (K & I-S), and simulated keratometry (average Sim K). However, there were no significant differences in keratoconus sensitivity between the classification network, KCI, and K & I-S. The sensitivity and specificity of average Sim K were significantly worse than those of the other tests. The classification network had significantly better sensitivity (P < 0.001) and specificity (P = 0.025) for KCS detection than the K & I-S., Conclusions: The neural networks completely distinguished KC from KCS and from topographies that resembled KC. The network approach equaled the sensitivity of currently used tests for keratoconus detection and outperformed them in terms of accuracy and specificity.

Purpose: Videokeratography is a powerful tool for the diagnosis of corneal shape abnormalities. However, interpretation of the topographic map is sometimes difficult, especially when pathologies with similar topographic patterns are suspected. The neural networks model, an artificial intelligence approach, was applied for automated pattern interpretation in corneal topography, and its usefulness was assessed., Methods: One hundred eighty-three topographic maps were selected and classified by human experts into seven categories: normal, with-the-rule astigmatism, keratoconus (mild, moderate, advanced), postphotorefractive keratectomy, and postkeratoplasty. The maps were divided into a training set (108 maps) and a test set (75 maps). For each map, 11 topography-characterizing indices calculated from the data provided by the TMS-1 videokeratoscope, plus the corresponding diagnosis category, were used to train a neural network., Results: The correct classification was achieved by a trained neural network for all 108 maps in the training set. In the test set, the neural network correctly classified 60 of 75 maps (80%). For every category, accuracy and specificity were greater than 90%, whereas sensitivity ranged from 44% to 100%., Conclusions: With further testing and refinement, the neural networks paradigm for computer-assisted interpretation or objective classification of videokeratography may become a useful tool to aid the clinician in the diagnosis of corneal topographic abnormalities.

Purpose: Videokeratography of early keratoconus may be difficult to distinguish from contact lens-induced corneal warpage, even by experienced examiners. Furthermore, topographic irregularity may be judged inconsistently if quantitative standards are not applied. Quantitative measures based on videokeratographic data were developed and evaluated to determine if improved corneal topographic classification can be achieved., Methods: The Corneal Irregularity Coefficient (CIC) and Corneal Power Coefficient (CPC) were derived from multiple measures of mean corneal power and its variance for 207 videokeratographs of normal, warped, keratoconus, and keratoconus-suspect corneas. CIC was plotted against CPC, creating a distribution of points representing all maps that tended to be grouped according to surface conditions (the Keratomorphic Diagram). Normal, steep, abnormal, and warped zones were defined by CIC and CPC cutoff values chosen to distinguish normal from keratoconus corneas graphically., Results: Seventy of 76 normal corneas were grouped in the normal zone and 6 in the steep zone; 84 of 84 keratoconus corneas were grouped in the abnormal zone; 35 of 35 contact lens-induced warpage cases were grouped in the warped zone; and 10 of 12 keratoconus-suspect corneas were grouped in the warped zone, with 2 in the abnormal zone. Serially plotted data of keratoconus progression and warpage regression demonstrated that the vector displacement of CIC and CPC values may provide a potentially useful means of distinguishing contact lens-induced warpage from keratoconus-suspect corneas., Conclusion: The Keratomorphic Diagram aids in classifying and comparing corneal shape by plotting indices along axes with easily recalled scales. The diagram may become a useful tool to assess presurgical corneal surface instability and postoperative progression of corneal shape change due to healing.

Purpose: Although visual inspection of corneal topography maps by trained experts can be powerful, this method is inherently subjective. Quantitative classification methods that can detect and classify abnormal topographic patterns would be useful. An automated system was developed to differentiate keratoconus patterns from other conditions using computer-assisted videokeratoscopy., Methods: This system combined a classification tree with a linear discriminant function derived from discriminant analysis of eight indices obtained from TMS-1 videokeratoscope data. One hundred corneas with a variety of diagnoses (keratoconus, normal, keratoplasty, epikeratophakia, excimer laser photorefractive keratectomy, radical keratotomy, contact lens-induced warpage, and others) were used for training, and a validation set of 100 additional corneas was used to evaluate the results., Results: In the training set, all 22 cases of clinically diagnosed keratoconus were detected with three-false-positive cases (sensitivity 100%, specificity 96%, and accuracy 97%). With the validation set, 25 out of 28 keratoconus cases were detected with one false-positive case, which was a transplanted cornea (sensitivity 89%, specificity 99%, and accuracy 96%)., Conclusions: This system can be used as a screening procedure to distinguish clinical keratoconus from other corneal topographies. This quantitative classification method may also aid in refining the clinical interpretation of topographic maps.

In the rabbit corneal stroma, the swelling pressure, P, has been reported to have an anomalous (negative) temperature coefficient, alpha P, contradicting traditional Donnan swelling theory. A parallel-plate, diffuse double layer Gouy-Chapman model was used to resolve this discrepancy. The present model incorporates the possibility that surface charge, sigma, is temperature dependent. It is shown that negative, zero, or positive coefficients of swelling pressure change with temperature are not mutually exclusive conditions, but can be attributed to the same underlying mechanism. For likely values of alpha P(range -7 x 10(-3) K-1 to +3.2 x 10(-3)K-1), the effective stromal charge has a negative temperature dependency, or dln sigma/dT less than 0. The present formalism is robust against variation in assumed alpha P, and is able to simultaneously satisfy the known values of swelling pressure, its thermal dependency, and stromal charge. These results implicate significant coulombic forces behind P. Predicted stromal surface charge is approximately 0.01 Cm-2. The predictions were confirmed with macrocontinuum Donnan swelling theory, suggesting that Donnan osmotic swelling is the principal macroscopic component of P.

An addition to the specular microscope is described which allows it to record the thickness of the excised cornea automatically as a function of time. The focus of the instrument is scanned mechanically through the tissue, and the position of the reflecting surfaces is detected by a photo-electric system and marked on a chart recorder. The system is able to follow thickness changes over periods of many hours and with an accuracy greater than obtainable by manual operation. This system has been helpful in the evaluation of a new medium which considerably extends the useful lifetime of the corneal endothelial fluid pump.

Evidence is presented that serotonin acts as a neurotransmitter in the cornea of the adult rabbit. Serotonin was localized to granules in a sparse population of subepithelial corneal nerves by an electron microscopic histochemical procedure. Significant endogenous levels of serotonin and its principal metabolite, 5-hydroxyindoleacetic acid, were detected in the central cornea by a fluorometric assay. Exogenous serotonin stimulated ion transport by corneal epithelium. This effect was potentiated by monoamine oxidase inhibition and was unaffected by an alpha-adrenergic receptor antagonist. Serotonin-stimulated ion transport was inhibited by the specific antagonist, methysergide, and by the replacement of Cl- with an impermeable anion. In tracer experiments, the serotonin-stimulated ion transport was shown to be caused by increased epithelial Cl- secretion. The serotonin response was partially inhibited by the beta-adrenergic antagonist, timolol. In a companion article, assay of corneal cyclic AMP showed stimulation of cyclic AMP synthesis by serotonin, inhibition by the specific antagonist, lysergic acid diethylamide, and potentiation by monoamine oxidase inhibition. We postulate that specific serotonergic receptors are present in the corneal epithelium and that activation of these receptors by serotonin released from serotonergic neurons increases the level of cyclic AMP, which stimulates active Cl- secretion by the corneal epithelium.

The addition of micromolar quantities of dopamine stimulated ion transport in the isolated rabbit corneal epithelium. This response was blocked by pretreatment with the dopamine antagonist, haloperidol, and by the elimination of Cl- from the bathing solutions. The beta-adrenergic antagonist, timolol, was also a potent inhibitor of the epithelial response to dopamine. The presence of the serotonin antagonist, methysergide, or the dopamine beta-hydroxylase inhibitor, FLA-63, did not significantly alter the corneal response to dopamine. Following superior cervical ganglionectomy, the epithelial response to dopamine was abolished. These findings are consistent with the idea that Cl- secretion in the rabbit corneal epithelium can be modulated by preterminal dopamine receptors located on the sympathetic nerve fibers; therefore, dopamine stimulation appears to be a serial process mediated by the release of norepinephrine from sympathetic nerve terminals in the epithelium.

The effects of several components of ophthalmic preparations on isolated rabbit cornea were studied by continuous electrophysiologic monitoring followed by fixation for scanning electron microscopy (SEM). Benzalkonium chloride (0.001 percent), thimerosal (0.0004 percent), and amphotericin B (0.0025 percent) all briefly increased ion transport, then greatly decreased epithelial resistance. Severe disruption of surface cell layers occurred simultaneously with resistance decrease. Silver nitrate (0.00017 percent) stimulated transport with less accompanying morphologic damage. Tetracaine (0.05 percent) disrupted epithelial function and caused exfoliation of several cell layers. Chlorobutanol (0.1 percent) produced a nearly complete loss of the squamous cell layer. Chloramphenicol, epinephrine, and pilocarpine produced minor changes in structure and electrophysiology at full clinical concentration. It was concluded that low concentrations of preservatives in ophthalmic preparations disrupt the barrier and transport properties of the corneal epithelium.

The cornea is dually innervated, receiving afferent nerves from the trigeminal ganglion and efferent nerves from the superior cervical ganglion. This study examines the specific effects of superior cervical ganglionectomy (SCGX) on the in vitro ion transport characteristics of the rabbit corneal epithelium. Two weeks after SCGX, epithelial Cl--dependent transport and total ionic conductance were increased in comparison to values obtained in paired control eyes. This increased transport level appeared to be independent of membrane receptor activity as demonstrated by lack of responsiveness to alpha-adrenergic, beta-adrenergic, serotonergic, dopaminergic, nicotinic cholinergic, or muscarinic cholinergic blockade. Nevertheless, SCGX produced a supersensitivity to epinephrine-stimulated transport as measured by the responsiveness of the ion transport current. Furthermore, SCGX abolished the responsiveness of the epithelium to serotonin. On the basis of these and earlier findings, the authors conclude that corneal sympathetic innervation influences membrane and receptor properties. Autonomic neurotrophic effects in the corneal epithelium include suppression of apical membrane Cl- permeability and of beta-adrenoreceptor sensitivity to biogenic amines. It is proposed that the corneal serotonergic receptors that activate Cl- transport lie on the sympathetic nerve terminals and stimulate this transport process by causing the neural release of a catecholamine.

1. Membrane events accompanying adrenaline-stimulated Cl secretion by the isolated rabbit corneal epithelium were investigated with micro-electrodes. 2. Pulses of adrenaline (5 X 10(-10) M final concentration) delivered to either side of the epithelium produced a transient decrease in epithelial resistance occuring at the outer membrane of the squamous cell. This response was reversible and could be blocked completely by total Cl substitution with SO4. 3. Adrenaline generally produced a small transient increase in epithelial potential occuring also at the squamous cell outer membrane. Reversal potentials obtained for the adrenaline response were 45-1 mV for corneal potential and 22-8 mV for outer membrane. 4. Adrenaline always hyperpolarized epithelial potential when the tear side was bathed in Cl-free solution. Reversing the gradient (Cl-free on the stromal side) slowly and consistently changed the response to a depolarization which reached a steady level after 2 hr. 5. The reversal potential of the outer membrane for the adrenaline response was found to be a semilogarithmic function of the tear side Cl concentration over a broad range with a slope of 56 mV/decade. The reversal potential was zero at a tear side Cl concentration of 41-5 mM, which value may be taken to be representative of cell Cl concentration. 6. After abolishing the adrenaline response by perfusing both sides of the tissue with Cl-free solution, reintroduction of Cl to the stromal side led to a recovery of the epithelial potential response in the hyperpolarizing direction. The recovery of the response was inhibited by ouabain (10(-5) M). 7. The results supported the following model for the influence of adrenaline on anion transport in the epithelium: Cl is transported against an electrochemical potential gradient into the cells from the stromal side by an active process linked to Na-k activated ATPase. Normally a slight gradient exists from cells to tears favouring the passive outward diffusion of Cl. This latter process is enhanged by adrenaline, which increases cell cyclic AMP, in turn increasing the passive Cl permeability of the outer cellular membrane.

Keratoscopy is a useful clinical tool for the evaluation of topographic abnormalities of the corneal surface. However, not all the detailed information presented by keratoscope photographs is assessed easily by visual inspection. A computer-based analysis system therefore was developed to assist in the clinical interpretation of keratoscope images. With this system, deviations from sphericity are displayed in graphic form to aid in the recognition of abnormalities, and surface powers are presented in tabular form. Human eyes that are emmetropic, and eyes with keratoconus and severe astigmatism were analyzed. This process provides a useful quantitative method with which to determine corneal shape, as well as a useful adjunct to the clinical evaluation and teaching of keratoscopy. In addition, such a quantitative analysis may provide the basis for the development of new techniques for the correction of visual distortion caused by corneal surface irregularities.

1. The mechanism underlying stromal oedema subsequent to epithelial hypoxia was investigated in isolated rabbit corneas. 2. Stromas swelled about 20 micrometer following a 1 hr period of tear side hypoxia in both whole corneal isolates and in preparations in which fluid movement across the endothelium was blocked with silicone oil. In the experiments using whole corneas, stromal thickness was independent of tear side oxygen tension as long as aqueous humour PO2 was greater than 40 mmHg. 3. Neither epithelial thickness nor epithelial electrical resistance, a measure of total ion permeability, was significantly affected by blocking respiration. 4. A 10 degrees C reduction in corneal temperature markedly reduced the rate of hypoxic swelling, suggesting the involvement of a metabolism-dependent hydrating process and implicating the stromal accumulation of a catabolyte. 5. When CN- was used to mimic the hypoxic effect in isolated whole corneas, the passive 36Cl unidirectional flux was unaffected, but lactate production rate and stromal [lactate] more than doubled. 6. These measurements were used with a mathematical model for corneal hydration dynamics to examine the causes of hypoxic oedema. The principal conclusions were: epithelial hypoxia enhances epithelial lactate production and release to the stroma; this process causes an increase in stromal lactate concentration and a decrease in stromal NaCl concentration (primarily through dilution); stromal lactate accumulation exceeds in osmotic load and dilutional effect on [NaCl], producing stromal oedema. Whereas hypoxia produces corneal metabolic acidosis, effects on endothelial permeability of HCO3- transport need not be postulated to explain the stromal oedema that results from hypoxia.

Swollen rabbit corneas incubated in vitro with their posterior surfaces blocked with silicone oil maintained fairly constant thickness over an 11 hr. period. Increasing the simulated intraocular pressure from 10 to 30 mm. Hg did not produce stromal thinning. When theophylline was added to stimulate epithelial Cl secretion by increasing the Cl permeability of the tear-facing epithelial membrane, corneas thinned at the average of 1.3 micrometer/hr. over a 6 1/2 hr. period. When the epithelial perfusion solution was made Cl-free by SO4 substitution to favor the passive flow of Cl from the cells to the tear solution, thinning of 3.91 micrometer/hr. over a 7 hr. period was observed. When corneas were perfused with Cl-free medium plus theophylline, thinning at the average rate of 6.20 micrometer/hr. over an 8 hr. period was achieved. Therefore the corneal epithelium is capable of thinning a swollen stroma by transport of fluid coupled to its Cl secretion, which can be enhanced by simple substitutions in the tear-side bathing solution.

The rapid and complete repair of the corneal epithelium following ocular surgery or trauma is essential for the maintenance of normal visual acuity. In this study the authors examined epithelial wound healing in the rabbit after cells were mechanically removed leaving the basal lamina intact. The decrease in wound area (mm2/hr) was neither linear nor amenable to simple kinetic analysis. However, analysis of the data in terms of the decrease in wound radius (mm/hr) revealed a biphasic process consisting of an initial latent phase with no epithelial movement (5.5 +/- .3 hr), followed by a linear healing phase. The rate of epithelial movement in the linear healing phase was 64 +/- 2 microns/hr. Neither the latent phase nor the rate of epithelial migration during the healing phase was affected by variations in initial wound size. Ultrastructural studies demonstrated that during the latent phase there was an increased desquamation of surface cells as well as cellular and subcellular reorganization of the basal cells. At the end of the latent phase, the leading edge of the wound was composed of a single cell layer. The onset of epithelial migration coincided with the first ultrastructural observation of typical ruffled membranes and filopodia. This work demonstrates that the analysis of the decrease in wound radius provides a straightforward and accurate means to assess the kinetics and therapeutic modulation of epithelial wound healing.

1. A quantitative basis for the currently accepted theory on the regulation of corneal hydration was derived using the technique of finite element analysis to integrate a set of coupled flow equations. The model was based on non-equilibrium thermodynamics and incorporated the transport and permeability properties of the corneal epithelium and endothelium as well as the gel properties of the central connective tissue layer. 2. Considerable errors were introduced in the prediction of corneal hydration dynamics (unsteady-state behaviour) unless allowance was made for the development of trans-stromal gradients in pressure and solute concentration. 3. Thickness of in vitro rabbit corneal epithelium and stroma were measured with an automatic specular microscope during responses to changes in the osmolarity of the tear-side bathing medium. The time course of these experiments was fitted with the mathematical model to obtain a set of membrane phenomenological coefficients and transport rates. 4. The model with the redetermined membrane parameters was tested by predicting the influence of other variations in boundary conditions with excellent match to several well-documented experimental observations, including an explanation for the slight stromal swelling observed in hibernating mammals. 5. The regulation of corneal stromal hydration can be explained accurately by balance between the dissipative flows across the serial array of corneal layers and the active HCO3 transport by the endothelium, supporting the earlier 'pump-leak' hypothesis. 6. It was found that stromal retardation of fluid flow, as well as gradients in solute concentration, significantly influences the dynamics of corneal stroma hydration. Tissue gel properties may be a more important factor in coupled transport across cell layers than generally appreciated.

Serotonin increases the level of cyclic AMP in incubated rabbit corneas; the concentration of agonist producing half-maximal stimulation is approximately 1.5 microM. Nialamide, an inhibitor of monoamine oxidase, potentiates the response to serotonin but not to epinephrine. Amitriptyline, an inhibitor of neuronal uptake of serotonin, does not potentiate the stimulation of cyclic AMP synthesis. Lysergic acid diethylamide, but not timolol, antagonizes the response to serotonin; the half-maximal inhibitory concentration is approximately 6 nM lysergic acid diethylamide. A comparison of the time course of the increase in cyclic AMP synthesis after addition of serotonin or epinephrine to the incubation media indicates that serotonin, but not epinephrine, must penetrate a barrier to its free diffusion. We conclude that the corneal epithelium contains specific serotonergic receptors that, upon activation, cause the synthesis of cyclic AMP, which mediates the stimulation of chloride transport (c.f. companion article, Klyce et al.). The serotonergic receptors must be at a location posterior to the beta-adrenergic receptors, which are on the anterior-surface of the apical cells.

1. The potentials and resistances associated with the cell membranes of the rabbit corneal epithelium were studied with 3 M-KCl-filled micro-electrodes.2. In the isolated cornea, the transepithelial potential was identical in polarity and magnitude to the simultaneously measured total corneal potential. In contrast to previous findings, the stromal potential was positive to the tear side. Negative stromal potentials apparently derive from inadequate electrodes or method of penetration, and were not found to be a function of filling solution. Transepithelial potential was also identical to over-all corneal potential in the living rabbit eye.3. In the isolated preparation, the average potential profile occurred in three distinct steps across the epithelium. By means of iontophoretic dye injection it was shown that these steps occurred across the outer membrane of the squamous cell, the transition region between the wing and basal cell, and across the inner membrane of the basal cell.4. The transverse membrane resistance of the outer epithelial membrane accounted for 60% of total corneal resistance. As a result, short-circuit current, which depolarizes the cornea, led to a hyperpolarization of the outer membrane, while affecting deeper membrane potentials little or not at all.5. The spontaneous potential of the outer membrane varied inversely with corneal potential in both normal and chloride-free Ringer, while the potential of the inner membrane of the basal cell was relatively constant, approaching the theoretical Nernst potential for potassium. The potential of the outer membrane was at chloride equilibrium and was sensitive to extracellular shunts. A Thevenin equivalent drawn for the epithelium suggested that half of the outer membrane potential could be attributed to loop currents. The potential step between wing and basal cells could be accounted for in terms of loop currents driven by the corneal potential through the epithelium.6. The potential profile of the frog corneal epithelium was similar to that of the rabbit. However, the major resistance in the frog cornea was associated with the basal cell membrane rather than with the squamous cell outer membrane. Quasi-instantaneous rectification was found for both epithelia. In the rabbit chloride rectified inwardly.