Edward Y. Zavala

Delayed Corneal Wound Healing Following Radial Keratotomy

Four human corneal specimens obtained 5 to 47 months following radial keratotomy were evaluated by correlative microscopy. Repeat radial keratotomies had been performed in two cases. We documented gaping keratotomy incisions, epithelial plugs, and epithelial-lined incisions. In all cases, Bowmans layer was malapposed with slight to moderate fibroblastic activity at the incision sites. Epithelial and endothelial radial ridges were seen in two cases. No endothelial damage was seen under the radial incisions. The morphological evaluation of these specimens show the potential for a poor wound-healing response when corneas with previous corneal surgery and/or pathologic states undergo radial keratotomy.

An Ultrastructural and Histochemical Study of Long-Term Wound Healing after Radial Keratotomy

Using correlative microscopy and plant lectins conjugated to fluorescent dyes, we studied two human corneal specimens obtained 66 and 70 months after radial keratotomy. In one case a second radial keratotomy had been performed 11 months before we obtained the corneal specimen, which allowed us to compare wounds 11 and 66 months old in the same specimen. The 11-month-old incisions demonstrated morphologic features consistent with incomplete wound healing, whereas the incisions examined 66 and 70 months postoperatively demonstrated complete wound healing. A different lectin binding pattern of wheat germ agglutinin and concanavalin-A was observed in the 11-month-old wounds, compared with the 66-month-old wounds. Published and unpublished morphologic studies of 17 human keratotomy specimens have demonstrated incomplete wound healing up to 47 months postoperatively. The present study documents complete corneal wound healing 66 months after radial keratotomy.

Hydrogel keratophakia in non-human primates

We performed sixteen keratophakia procedures in nine non-human primates using various hydrogel lenses (38% to 79% water) as donor corneas (lenticules). The lenses were treated as if they were donor human corneas using currently practiced keratophakia procedures including microkeratome resection of the recipient cornea, staining of the lenses before cryolathing and, in some cases, lathing according to a modified computer program after which the lenses were inserted into a previously dissected recipient lamellar pocket. This technique was able to produce steepening of the cornea up to 44.8 diopters and was well tolerated for over three months. The use of hydrogel lenses could potentially eliminate many of the negative aspects of the use of human donor corneas for the surgical correction of aphakia.

Endothelial Cell Density Determined by Specular Microscopy and Scanning Electron Microscopy

Human eyes were photographed with a specular microscope and later examined wit a scanning electron microscope. Corneas from patients undergoing corneal transplantation in whom we were able to obtain preoperative specular micrographs were similarly analyzed. An attempt was made to correlate the counts obtained with both microscopic techniques by determining the amount of shrinkage the cornea undergoes while being processed for SEM. All specimens were counted with a planimeter. We found that the specular microscope adequately analyzes the endothelial cell density in the central and paracentral cornea of a normal eye, but because of its small sampling area specular microscopic counts are subject to significant error when dealing with nonhomogeneous populations such as postoperative cases. We found the peripheral corneal endothelial density to be less than the central endothelial density. Furthermore, we found that we could maximize the accuracy of counting by using a variable frame in a nonhomogeneous population, counting a minimum of four photographs per specimen, analyzing different areas, and analyzing larger areas.

Correlative Microscopy and Tissue Culture of Congenital Hereditary Endothelial Dystrophy

A 4 1/2-year-old boy with congenital hereditary endothelial dystrophy underwent successful bilateral penetrating keratoplasties. Visual acuity in both eyes was restored to 6/18 (20/60). Correlative microscopic analysis of the corneal button obtained from the first transplant showed an irregular epithelium, a continuously thickened Bowmans layer, fragmentation and homogenization of the anterior stromal lamellae, a mildly enlarged stromal fibril diameter, abnormal collagen layers posterior to Descemets membrane, atrophic paracentral corneal endothelium, and absent central endothelium. The endothelial cells from the second corneal button were placed in tissue culture and failed to demonstrate any growth characteristics of differentiation into normal appearing cells after 14 days. We concluded that the endothelial cells in congenital hereditary endothelial dystrophy are functionally and morphologically abnormal. Early penetrating keratoplasty can be successful in such cases.

Alloplastic Implants for the Correction of Refractive Errors

Convex-shaped, hydrophilic lenticles of various water content were implanted into microkeratome-dissected corneal pockets in a baboon model. Freshly trephined, cryolathed, and factory-lathed hydrogels were evaluated for periods exceeding 15 months, documenting clinical response, corneal curvature shifts, and morphologic features. Previously implanted hydrogels were subsequently removed, documenting the reversibility of the procedure while allowing the corneas to return to their preoperative condition. Various alloplastic materials were then reimplanted into the same intrastromal pockets. Medium and high water content lenses produced good results without affecting the recipient cornea, whereas thick or low water content materials extruded or produced severe anterior segment inflammation. The use of medium and high water content, convex-shaped lenticles for the correction of aphakia is possible using current techniques.

Clinical pathology of non-freeze lamellar refractive keratoplasty.

We studied two corneal specimens obtained following corneal transplantation for loss of best corrected vision after planar lamellar refractive keratoplasty. The epithelium appeared slightly undifferentiated in both cases. Peripheral bends and occasional breaks were found in the periphery of Bowmans layer. In one case there were areas of subepithelial fibrosis as well as ultrastructural fractures in Bowmans layer. The keratocyte population appeared to be slightly decreased in one case. In both specimens the optical interface contained active fibroblasts in the periphery. The planar lamellar refractive keratoplasty technique theoretically eliminates many of the adverse morphologic features encountered following the standard Barraquer cryolathe techniques, but clinical studies will be needed to determine if this form of lamellar keratoplasty is clinically superior to currently practiced techniques.

Refractive keratoplasty: Myopic keratomileusis in baboons

We performed 8 myopic keratomileusis procedures in a baboon (Papio cynocephalus) model using the same instruments and techniques which are presently clinically employed. We requested 10 diopters of refractive correction and were able to achieve a mean keratometric correction of 5.5 diopters (range 2.4-8.4) in 6 eyes. Keratometric data in the two remaining eyes was not obtained, because of distortion of corneascope photographs. We did not encounter any technical problems at surgery or during the 2-1/2 to 6 month postoperative period of followup. Morphologic analysis demonstrated subclinical, focal breaks in Bowmans membrane in all specimens and keratocyte repopulation in 7 of 8 eyes. We did find a wide range in the central thickness of the excised corneas which can account for the undercorrections achieved. We consider the baboon model to be excellent for the evaluation of myopic keratomileusis.

Epikeratophakia: clinical evaluation and histopathology of a non-human primate model.

We performed four aphakic and two myopic epikeratophakia procedures in a non-human primate model using two fresh and three prefrozen corneas and one glycerin-preserved cornea. The experiments were terminated 4 to 5 months postoperatively, and the eyes examined with correlative microscopy. The glycerin-preserved cornea sloughed and was not available for analysis. All but one prefrozen specimen demonstrated donor keratocyte repopulation. Active keratocytes were noted lining the optical interface. Focal areas of missing Bowmans layer were present in three recipient corneas. Epithelial interface ingrowth occurred in two specimens. The recipient corneas were otherwise normal.

Keratocyte attachment to hydrogel materials

We have developed an in-vitro model for keratocyte interactions with hydrogel materials. With the aid of inverted phase contrast microscopy and scanning electron microscopy, we observed the growth of baboon stromal keratocytes on the surface of various hydrogel materials. In this study, the Bioflex lens (0.5% hydration) surface was extensively covered with keratocytes, whereas 1% to 25% of the surfaces of Crofilcon A (40% hydration) and Perfilcon A (70% hydration) lenses were covered with keratocytes. The Bufilcon A (55% hydration) lens was free of keratocyte attachment. Keratocytes attached to the surfaces of the lenses produced extracellular collagenous material as evidenced by positive staining with alcian green-metanil yellow. These in-vitro results correlate with our non-human primate studies using Bioflex, Crofilcon A and Bufilcon A lenses as intracorneal implants.