Synthesis and properties of regenerated cellulose-based hydrogels with high strength and transparency for potential use as an ocular bandage.

Cellulose is a biologically derived material with excellent wound-healing properties. The high strength of cellulose fibers and the ability to synthesize gels with high optical transparency make these materials suitable for ocular applications. In this study, cellulose materials derived from wood pulp, cotton, and bacterial sources were dissolved in lithium chloride/N,N-dimethylacetamide to form regenerated cellulose hydrogels. Material properties of the resulting hydrogels, including water content, optical transparency, and tensile and tear strengths, were evaluated. Synthesis parameters, including activation time, dissolution time, relative humidity, and cellulose concentration, were found to impact the material properties of the resulting hydrogels. Overnight activation time improves the optical transparency of the hydrogels from 77% to 97% at 550 nm, whereas controlling cellulose concentration improves their tear strength by as much as 200%. On the basis of the measured transmittance and strength values of the regenerated hydrogels prepared via the optimized synthesis parameters, Avicel PH 101, Sigma-Aldrich microcrystalline cellulose 435236, and bacterial cellulose types were prioritized for future biocompatibility testing and potential clinical investigation.

Mitomycin C reduces corneal light scattering after excimer keratectomy.

PURPOSE: To evaluate the effect of intraoperative mitomycin C (MMC) on corneal light scattering after excimer laser keratectomy. METHODS: Phototherapeutic keratectomy (PTK) was performed in 24 rabbit eyes. After 40-microm epithelial ablation, animals were divided into three groups. In group 1, filter paper discs soaked with MMC (group 1A, 0.5 mg/mL; group 1B, 0.25 mg/ml) were applied for 1 minute. In group 2, annular filter papers soaked with MMC (group 2A, 0.5 mg/mL; group 2B, 0.25 mg/mL) were applied for 1 minute. Controls received vehicle only (group 3). Six-millimeter diameter 100-microm deep PTK was performed. Corneal light scattering was measured weekly from 1 to 6 weeks, at 10 weeks, and at 8 and 13 months using a scatterometer. A corneal light scattering index (SI) ranging from 0 to 10 was calculated; SI of 1 represents normal scattering. RESULTS: A statistically significant decrease in mean SI was noted in group 2A (annular MMC 0.5 mg/mL; p<0.05) as compared with the control group at 2 weeks. At 10 weeks, SI approached baseline levels in group 2 and the control group but showed significant increase in group 1 (MMC disc; p < 0.05). At 8 and 13 months, SI showed no statistical differences between groups. CONCLUSIONS: Controlled application of 0.5 mg/mL MMC in the corneal midperiphery transiently reduces corneal light scattering after excimer keratectomy in this rabbit model.

Epithelial damage thresholds for multiple-pulse exposures to 80 ns pulses of CO2 laser radiation.

Corneal epithelial damage thresholds for exposures to 80 ns pulses of 10.6 microm infrared radiation produced by a CO2-TEA laser were investigated. Thresholds were determined for sequences of 1, 2, 8, 32, 128, and 1,024 pulses at pulse repetition frequencies of 10 and 16 Hz. Threshold damage is correlated by an empirical power law of the form EDth = CN(-alpha) in which EDth, is the threshold radiant exposure per pulse, and N is the number of pulses. The constants C and alpha are similar for the two pulse repetition frequencies. For the combined data set, C = 2,955 J m(-2) pulse(-1) (295.5 mJ cm(-2) pulse(-1) and alpha = 0.178. This value of the constant C is within 5% of the measured damage threshold for a single 80 ns pulse exposure. Temperature calculations reveal that the maximum temperature increase on the beam axis, 10 microm beneath the anterior tear surface, resulting from the different threshold exposures is constant to within +/-10% of the mean values. This result is consistent with a critical temperature damage model. Damage threshold measurements on cooled corneas indicate that the damage mechanism indeed has a substantial thermal component.

Velocity measurements of normal and sickle red blood cells in the rat retinal and choroidal vasculatures.

The purpose of this study was to develop an in vivo, noninvasive method to assess the velocities of normal and sickle red blood cells (RBCs) in the retinal and choroidal vasculatures of rats. Human and rat RBCs were isolated from whole blood, labeled with fluorescein isothiocyanate (FITC), and administered intravenously to anesthetized rats. A Rodenstock scanning laser ophthalmoscope (SLO) was used to image the FITC-labeled RBCs as an NTSC video signal. Video sequences of RBC transit in the retinal (pigmented rats) and choroidal (albino rats) vessels were captured directly to digital format. Following in vivo angiography, the animals were sacrificed, the eyes enucleated, and retinas prepared by our adenosine diphosphatase vascular labeling technique for viewing by conventional optical microscopy. Although rat and normal human RBCs differ slightly in size, their velocities were similar in the retinal arteries and capillaries (within 4%). Velocities of RBCs from sickle cell patients (sRBCs) were slower by 12 and 9% in arteries and by 38 and 25% in capillaries, compared to rat and normal human RBCs, respectively. Compared to velocities in retinal capillaries, the velocities in choroidal capillaries were much slower for rat RBCs (77%), normal human RBCs (79%), and sRBCs (67%). In contrast to normal human RBCs, sRBCs were often retained transiently in retinal capillaries at preferred sites, but in choroidal capillaries large numbers of cells were retained for extended periods. SLO imaging of FITC-labeled RBCs in rat retina and choroid provided a reliable method for evaluating normal and abnormal hemodynamics.

Small-angle light scattering and birefringence properties of chick cornea.

PURPOSE: Techniques employing polarized light propagation and scattering are useful in examining the cornea's lamellar structure. Recent advances in theoretical methods have significantly increased the ability to relate features of lamellar arrangements to measurements of transmitted polarized light. The chick cornea, because of its hypothesized structure of a gradual helical rotation of lamellar pairs, presents an interesting model for further development of this methodology. METHODS: Small-angle light scattering (SALS) and polarized transmission measurements were made on 7-week-old chick corneas under conditions that closely approximate the physiological state. Birefringence properties were determined from the transmission measurements and compared to the results of model calculations of polarized light propagating through lamellae organized according to the hypothesized structure for chick cornea. RESULTS: The I+ small-angle light scattering pattern had 4 cloverleaf lobes aligned with the crossed polarizer and analyzer axes. The lobes disappeared when the transcorneal pressure was increased from zero to 18 mmHg. Retardation measured at 18 mmHg was very small (approximately 0.01 microm). CONCLUSION: The disappearance of the I+ small-angle light scattering pattern when IOP is increased suggests that the lamellae undulate in their relaxed state and the undulations straighten when IOP is increased. Measured birefringence properties are consistent with the hypothesized lamellar structure.

Polarized light propagation in corneal lamellae.

The propagation of polarized light through the cornea is affected by the orientations of the corneal lamellae and by the refractive imbalance between the collagen fibrils and the ground substance. Thus, well-designed measurements and analyses of polarized light propagation through the cornea can be used to obtain information regarding the cornea's lamellar and fibrillar structures. This paper shows that, for the rabbit, measured values of the optical parameters strongly suggest that the distribution of lamellae orientations is not random, but has one (or two) preferred orientation directions. Also, there is considerable evidence that collagen is intrinsically anisotropic. The Weiner formula gives the effective birefringence of an assembly of parallel isotropic fibrils and its generalization to the case of anisotropic fibrils is presented. Finally, calculations based on preferred orientation models having lamellae composed of anisotropic fibrils show that comparison with experimental values can yield structural information.

Spatial mapping of polarized light transmission in the central rabbit cornea.

In this paper polarized light transmission measurements are made under conditions that closely approximate the physiological state in order to probe lamellar structure in the central cornea of New Zealand white rabbits. The results are interpreted with the aid of a newly developed theory (published elsewhere) in which the cornea is modeled as stacked birefringent layers corresponding to the lamellae. The theory enables predictions of the statistical properties of lamellar ordering based on characteristics of the transmission of polarized light. The experimental results are consistent with a structure in which a number of lamellae have a fixed azimuthal orientation (i.e. about an axis normal to the corneal surface), whereas the remainder are essentially randomly oriented. Comparisons with the theoretical predictions are consistent with a model in which the preferred direction in the apical region of the cornea does not vary significantly among rabbits; and the preferred lamellar orientation direction determined for the population measured here is very close to that suggested in previous experiments on a smaller number of rabbits. Mapping experiments using a new goniometric holder showed that the preferential order at the apical region is closely preserved throughout the central approximately 4 mm diameter optical zone in individual corneas.

A numerical test of the normal incidence uniaxial model of corneal birefringence.

The suggestion that the central cornea can be modeled as a uniaxial birefringent material with its optic axis normal to the surface is explicitly tested by numerical calculations. A theoretical framework is presented to model the corneal stroma as a series of stacked, uniaxial birefringent layers (lamellae). Calculations are then made of the transmission of normally incident, linearly polarized light through model systems having various azimuthal orientations of the layers, motivated by the suggestion of an overall "random" organization of the stromal lamellae. It is concluded that the uniaxial description, and the assumptions upon which that description is based, do not hold for the cornea. In particular, the calculations are in agreement with recent experiments in which one always observes a non-zero cross-polarized transmission (hence birefringence) at normal incidence.

Objective measurement of corneal light scattering after excimer laser keratectomy.

PURPOSE: To obtain objective measurements of corneal light scattering after excimer laser keratectomy, and to evaluate the relation of light scattering to clinical haze grading and visual acuity. METHODS: The authors measured best-corrected visual acuity, subjective clinical haze grade, and corneal light-scattering index in patients undergoing photorefractive keratectomy (PRK) (n=26), and phototherapeutic keratectomy (n=8), preoperatively and postoperatively at 1,3,6,9, and 12 months or later. Corneal light scattering was correlated with visual acuity and clinical haze grade. RESULTS: Corneal light scattering increased after PRK and was reduced after phototherapeutic keratectomy. Corneal light scattering index showed a stronger positive correlation with logMAR visual acuity (r=0.57) than clinical haze grading (r=0.34). Corneal light-scattering index (P<0.05 at 1 and 3 months) and clinical haze grading (P<0.05 at 6, 9, and 12 months) were significantly higher in eyes undergoing PRK with ablation depths of more than 80 microns. CONCLUSIONS: Excimer laser surgery affects corneal light scattering. Ablations with depths greater than 80 microns produce significantly higher levels of haze than those less than 80 microns. Objective measurement of corneal light scattering may be useful in monitoring the outcome of excimer keratectomy.

Numerical modeling of the cornea's lamellar structure and birefringence properties.

A model of the cornea's lamellar structure is proposed that is capable of explaining experimental results obtained for the transmission of normal-incidence polarized light through rabbit and bovine cornea. The model consists of a large number of planar lamellae, each approximated as a uniaxial birefringent layer, stacked one upon another with various angular orientations. Polarized light transmission through the composite system is modeled theoretically by use of the Jones matrix formalism. The light transmission is calculated numerically for a large number of model lamellae arrangements, each generated from a statistical description, and histograms are constructed of various properties of the light transmission, including the minimum and maximum cross-polarized output intensities. It is demonstrated that various structural and optical parameters of the lamellae arrangements of actual corneas may be estimated by comparison of the calculations with detailed experimental data. Certain characteristics of the histograms are identified that permit a clear distinction between random and partially ordered systems. Comparisons with previously published experimental data provide strong evidence that the lamellae orientations are not entirely random, but rather a significant fraction are oriented in a fixed, preferred direction.

Ultrastructure in anterior and posterior stroma of perfused human and rabbit corneas. Relation to transparency.

PURPOSE: The authors sought to discover whether there are differences in the degree of spatial order in the fibrillar ultrastructure between anterior and posterior stroma. METHODS: Human corneas were obtained from eye bank eyes. Although they had been classified as normal, some swelling remained after 3 hours of deturgescence. Freshly excised, unswollen rabbit corneas also were used. Image analysis methods were applied to transmission electron micrographs of the anterior, middle, and posterior stroma of these corneas to determine the positions and radii of fibrils, the fraction of total area occupied by fibrils, and the fibril number density. Results were used to calculate the interference factor that appears in the direct summation of the fields for light scattering theory and to estimate the total scattering cross-section per fibril. The interference factor is a measure of the spatial order in the positions and sizes of the fibrils. RESULTS: Electron micrographs showed anterior-posterior variations in size and number density of fibrils. The interference factor at wavelengths of visible light was lower in posterior stroma than in anterior stroma for humans and rabbits. In some instances in humans, the anterior interference factor was characteristic of mildly swollen cornea. When averaged for the electron micrographs analyzed, the anterior stroma was predicted to scatter approximately twice as much light per unit depth as the posterior stroma in humans (at any given wavelength) and approximately three times as much in rabbits. CONCLUSIONS: Calculations of the interference factor showed that there were differences in the anterior-posterior spatial ordering of fibrils. In human corneas, the differences could have been caused by intrinsic in vivo differences between anterior and posterior stroma; however, possible anterior-posterior variations in swelling between the two regions in vitro also could have affected the results.

Antioxidants reduce corneal light scattering after excimer keratectomy in rabbits.

BACKGROUND AND OBJECTIVE: Excimer laser-corneal tissue interaction liberates highly reactive free radicals. Our aim was to determine if intraoperative application of antioxidants reduces the postoperative corneal light scattering by minimizing free radical-mediated cellular injury. STUDY DESIGN/MATERIALS AND METHODS: Phototherapeutic keratectomy (PTK) was performed in 20 rabbit eyes. Following 40-microns epithelial ablation, rabbits were divided into two groups and received 1-minute intraoperative application of either 50% dimethyl sulfoxide and 1% superoxide dismutase or vehicle (balanced salt solution) only. This was followed by a 6-mm diameter 100-microns PTK. Corneal light scattering was measured for 18 weeks using scatterometry. A light scattering index (SI) ranging from 0-10 was calculated; SI = 1 represents normal scattering. Light microscopic examination was performed in selected corneas. RESULTS: The mean baseline SI was similar for both groups (P = 0.95). Corneal haze followed a pattern of gradually increasing to peak at 2-3 weeks, and regressing partially thereafter. The decrease in mean SI following antioxidant application (dimethyl sulfoxide and superoxide dismutase) as compared to control group approached significance at 3 weeks (0.1 > P > 0.05) and was highly significant at 9 weeks (P < 0.05). CONCLUSION: Intraoperative application of antioxidants decreases light scattering following excimer keratectomy in rabbit corneas. UV-induced free radicals may play a role in the pathogenesis of corneal light scattering following excimer laser keratectomy.

Image processing of electron micrographs for light scattering calculations.

Theoretical light scattering calculations, when applied to models based on structures seen in electron micrographs of corneal stroma, require knowledge of the constituent fibril positions and radii. Obtaining this information manually is a difficult and time-consuming task. In order to facilitate this problem, we present a simple and flexible computer algorithm that allows the process to be automated using a Macintosh computer. The accuracy of the results is checked by comparing light scattering calculations using fibril positions and radii found manually with those found by the computer. The results show that the computer algorithm is a viable and accurate means of obtaining the needed data.

Cornea epithelial damage thresholds in rabbits exposed to Tm:YAG laser radiation at 2.02 microns.

We have determined exposure conditions for minimal damage to the corneal epithelium in rabbit using a continuous wave Tm: YAG laser operating in the TEM00 mode at incident powers between 80 and 450 mW. The 1/e beam diameter was 0.94 mm and exposure durations for threshold damage ranged from 4.3 to 0.08 sec. Calculated temperature increases on the beam axis 10 microns beneath the surface at the measured thresholds were essentially constant and averaged 44 degrees C. This is basically the same temperature increase found for threshold CO2 laser damage and suggests that the critical temperature damage model, which correlates CO2 laser damage, can predict damage thresholds for mid-infrared laser radiation. We also showed that reliable thresholds can be determined in freshly enucleated eyes, thus opening up the possibility of using available laser sources in laboratories not equipped and approved for animal experiments to determine damage thresholds.

Epithelial damage in rabbit corneas exposed to CO2 laser radiation.

Corneal injury thresholds are determined for conditions not previously explored for CO2 laser radiation, including multiple-pulse exposures and a systematic investigation of the effect of beam diameter on single-pulse damage thresholds. Multiple-pulse exposures from pulse trains up to 999 pulses, having pulse repetition frequencies between 1 and 100 Hz and individual pulse durations between 10(-3) and 0.5 s, were explored. Damage thresholds are discussed in terms of an approximate critical temperature model, the damage integral model and other empirical correlations. Single-pulse exposures are accurately correlated by an empirical critical temperature model in which the critical temperatures have a weak dependence on exposure duration. However, certain aspects of the single-pulse damage data led us to propose a new thermal damage model that incorporates an endothermic phase transition as the damage mechanism. This physical model accurately correlates single-pulse damage for exposures between 10(-3) and approximately 10 s.

Effects of fibril orientations on light scattering in the cornea.

It is generally assumed that the collagen fibrils in the stroma are the primary scatters of light in the nearly transparent cornea of the eye. We derive a scaling relationship between scattering angle and light wavelength that should hold if this hypothesis is valid. The derivation accounts for the cornea's layered nature and the azimuthal orientations of the fibrils in the different layers. The fibrils are treated as finite-length cylinders, and the scaling relation is obtained in both the far- and the intermediate-field zones. The predicted relationship is verified experimentally for normal-thickness rabbit corneas.

Stromal damage in rabbit corneas exposed to CO2 laser radiation.

Threshold damage to the cornea from CO2 lasers is confined to the epithelium. Exposures well above the threshold for epithelial damage produce bowl-shaped stromal wounds. Light and electron microscopy and slit-lamp photographs all show a sharp demarcation between the damaged and undamaged regions 48 hr after exposure. The micrographs also show that the damaged region is accellular. Calculations of the expected temperature increases combined with analyses of slit lamp photographs show that the wound boundary corresponds to a surface of equal peak temperature increase. Comparisons with epithelial and endothelial damage conditions suggest that stromal, endothelial and epithelial cells have essentially the same thermal damage mechanism.