Improving the success rate of human corneal endothelial cell cultures from single donor corneas with stabilization medium.

Main objective of this study was to improve the success rate of human corneal endothelial cell (hCEC) cultures from single donor corneas. We could show that the use of stabilization medium prior to cell isolation may have a positive effect on the success rate of hCEC cultures from single research-grade donor corneas by allowing growth of otherwise possibly not successful cultures and by improving their proliferative rate. hCEC were obtained from corneo-scleral rims of 7 discarded human research-grade cornea pairs. The Descemet membrane-endothelium (DM-EC) sheets of each pair were assigned to 2 experimental conditions: (1) immediate cell isolation after peeling, and (2) storage of the DM-EC sheet in a growth factor-depleted culture medium (i.e. stabilization medium) for up to 6 days prior to cell isolation. hCEC isolated by enzymatic digestion were then induced to proliferate on pre-coated culture plates. The success rate of primary cultures established from single donor corneas were higher for DM-EC sheets kept in stabilization medium before cell isolation. All cultures (7/7) initiated from stabilized DM-EC sheets were able to proliferate up to the third passage, while only 4 out of 7 cultures initiated from freshly peeled DM-EC sheets reached the third passage. In addition, for the 4 successful paired cultures we observed a faster growth rate if the DM-EC sheet was pre-stabilized prior to cell isolation (13.8 ± 1.8 vs 18.5 ± 1.5 days, P < 0.05). Expression of the phenotypical markers Na+/K+-ATPase and ZO-1 could be shown for the stabilized cultures that successfully proliferated up to the third passage.

Spontaneous corneal clearance after graft detachment in DMEK.

Descemet Membrane detachment is a potential complication after Descemet Membrane Endothelial Keratoplasty (DMEK). Here, we present a unique case of a DMEK surgery in a complicated eye that suffered a nearly complete DMEK graft detachment and later a graft opacification with a pseudo-anterior chamber. In Mid-November 2020, a planned DMEK was performed in a 64-year-old male patient due to corneal decompensation. Four months after DMEK, a fibrotic DMEK graft was seen across the anterior chamber with a pseudo-anterior chamber; however, the recipient cornea showed complete clearance with an endothelial cell count of about 1204 cells/mm2 and a best-corrected visual acuity of 20/25. Three months later, we observed a significant opacification of the detached graft, and the best-corrected distance visual acuity decreased to 20/63. We proceeded with the graft removal without performing a second DMEK. Ten months after graft removal, the cornea remained clear with an endothelial cell count of about 510 cells/mm2, and the best-corrected visual acuity was 20/25.

[Descemet membrane endothelial keratoplasty (DMEK): two-year results]. / Queratoplastia endotelial de membrana de Descemet (DMEK): resultados a dos años.

PURPOSE: To report the two-year results of Descemet membrane endothelial keratoplasty (DMEK) for managing corneal endothelial disorders. METHODS: Non-randomized prospective clinical trial. A DMEK was performed in ten patients with Fuchs' endothelial dystrophy or bullous keratopathy. A 3.5 mm clear corneal incision was made and "under air" DM was stripped off from the posterior stroma. A 9.0 mm diameter, organ cultured donor DM roll was inserted into a recipient anterior chamber, positioned into the posterior stroma and secured by completely filling the anterior chamber with air for 30 minutes. RESULTS: Three eyes showed complete detachment of the tissue; this was managed by a secondary Descemet stripping endothelial keratoplasty procedure. The remaining seven eyes had a best corrected visual acuity of >or=0.7 in three eyes (43%) at one month, in five eyes (71%) at six months, and in six eyes (86%) at one and two years. At six months, the endothelial cell density averaged 2039 (+/-373) cells/mm2 (n=7), at one year 1925 (+/-267) cells/mm2 (n=7) and at two years 1730 (+/-400) cells/mm2 (n=6). CONCLUSIONS: DMEK may provide quick and nearly complete visual rehabilitation. Since the donor tissue can be stripped from donor corneo-scleral rims, the procedure may be readily accessible to most corneal surgeons.

[Lamellar keratoplasty. Back to the future?!]. / Lamelläre Keratoplastik. Zurück in die Zukunft?!

In 1840 Mühlbauer was the first to describe a technique for anterior lamellar keratoplasty (LKP). However, in the second half of the twentieth century penetrating keratoplasty (PKP) became the gold standard. Although it is associated with a higher risk for serious complications -- it is technically easier to perform and avoids wound healing reactions in the lamellar interface and thus resulting in better visual acuity. In view of the pathology, replacing all layers of the cornea including healthy parts can be considered therapeutic "overkill" for many corneal graft indications. Several innovative surgical techniques have recently been described which allow the lamellar dissection of recipient and donor cornea with good reproducibility in almost every desired depth. This now allows the recipient endothelium and Descemet's membrane to be selectively replaced or preserved and to avoid formation of an optical barrier in the lamellar interface in eyes undergoing lamellar keratoplasty for optical indications. The most important principal advantage of an anterior LKP -- to minimize the risk of an immune reaction in the graft -- is even more important in tectonic indications. From the large number of variations, the surgical technique, results, and problems with anterior and posterior LKP for optical indications as well as lamellar segment keratoplasty and epikeratoplasty for tectonic indications are discussed.

Standardization of the Descemet membrane endothelial keratoplasty technique: Outcomes of the first 450 consecutive cases.

OBJECTIVES: To evaluate the clinical outcome of the first 450 consecutive cases after Descemet membrane endothelial keratoplasty (DMEK), as well as the effect of standardization of the technique. MATERIAL AND METHODS: Comparison between 3 groups: Group I: (cases 1-125), as the extended learning curve; Group II: (cases 126-250), transition to technique standardization; Group III: (cases 251-450), surgery with standardized technique. Best corrected visual acuity, endothelial cell density, pachymetry and intra- and postoperative complications were evaluated before, and 1, 3 and 6 months after DMEK. RESULTS: At 6 months after surgery, 79% of eyes reached a best corrected visual acuity of≥0.8 and 43%≥1.0. Mean preoperative endothelial cell density was 2,530±220 cells/mm2 and 1,613±495 at 6 months after surgery. Mean pachymetry measured 668±92 µm and 526±46 µm pre- and (6 months) postoperatively, respectively. There were no significant differences in best corrected visual acuity, endothelial cell density and pachymetry between the 3 groups (P > .05). Graft detachment presented in 17.3% of the eyes. The detachment rate declined from 24% to 12%, and the rate of secondary surgeries from 9.6% to 3.5%, from group I to III respectively. CONCLUSIONS: Visual outcomes and endothelial cell density after DMEK are independent of the technique standardization. However, technique standardization may have contributed to a lower graft detachment rate and a relatively low number of secondary interventions required. As such, DMEK may become the first choice of treatment in corneal endothelial disease.

A comparison of wound healing in sutured and unsutured corneal wounds.

We compared corneal wound healing in 25 clear transplants with 26 keratotomy specimens and sutured and unsutured corneal wounds in a monkey model. Monkey wounds healed faster than human wounds, but healing time within the same species varied. Sutured wounds were characterized by subepithelial fibroplasia, recovery of collagen fiber continuity and absence of epithelial plugs. Unsutured wounds had no subepithelial fibroplasia but had fibroblasts and collagen fiber orientation parallel to wounds. Unsutured wounds in corneas containing sutures had subepithelial fibroplasia, and fibroblast orientation and fiber deposition parallel to the wound. Epithelial ingrowth and incarceration of Bowman's layer and/or Descemet's membrane in unsutured wounds appeared to disrupt wound healing. Abnormalities in wound healing in sutured or unsutured wounds are associated with lamellar distortion, a modified inflammatory response, and individual wound healing responses.

Variation in healing throughout the depth of long-term, unsutured, corneal wounds in human autopsy specimens and monkeys.

OBJECTIVE: To determine regional differences in stromal wound healing morphologic features in long-term radial keratotomy wounds. METHODS: Unsutured, semiradial, nonperforating wounds in seven monkeys and 10 human autopsy specimens obtained 3.5 to 52 months after surgery were analyzed using light, transmission, and fluorescent microscopy. RESULTS: Monkeys showed fast healing, whereas all human specimens had one or more incisions containing a superficial epithelial plug. Completely healed wounds in both models had fibroblast orientation and ultrastructural "pseudolamellar" (ie, colinear collagen fiber) continuity across the anterior wound, compared with disorganization of the midposterior scar. CONCLUSIONS: Regional differences in healing rates, mechanical factors associated with epithelial plug elimination, intrinsic tissue characteristics, and/or induction of pseudolamellar repair by the epithelial plug may explain variation in healing within an unsutured corneal wound.

Epithelial-stromal interactions in human keratotomy wound healing.

OBJECTIVE: To evaluate epithelial-stromal interactions in the healing of stromal wounds and the relationship of such interactions to regional variations in healing throughout keratotomy wounds. METHODS: Ten radial keratotomy autopsy specimens were studied by using light and transmission electron microscopy. RESULTS: Underneath epithelial plugs, the epithelial-stromal interface was characterized by three adjacent morphological zones: a duplicated basement membrane complex, a zone that resembled Bowman's layer, and a third zone with collagenous fiber orientation parallel to the plugs. Scar tissue orientation was transverse at the base of the plug, and sagittal in deeper wound regions. CONCLUSIONS: Basement membrane duplication and a Bowman's layer-like region underneath a plug may result from complicated epithelial-stromal interaction. Asymmetrical organization of the scar, with subepithelial transverse and sagittal deeper scar tissue orientation, may characterize radial keratotomy wound healing, and may relate to variations in final refractive effect.

Posterior lamellar keratoplasty for a case of pseudophakic bullous keratopathy.

PURPOSE: To describe a new surgical technique for posterior corneal transplantation. METHODS: An elderly patient had painful, pseudophakic bullous keratopathy with low visual potential. Through a 9.0-mm scleral tunnel incision, a midstromal pocket was dissected across the cornea, and a posterior lamellar disk 7.0-mm in diameter, which consisted of posterior stroma, Descemet membrane, and endothelium, was excised. A similarly shaped donor posterior disk was implanted in the recipient opening without suture fixation, and the scleral incision was sutured. RESULTS: Throughout the postoperative period, the posterior corneal transplant remained clear and in position. Three months after surgery, the "suture-in" astigmatic error was 3.5 diopters. Pachymetry measured 0.44 mm. CONCLUSION: Posterior lamellar keratoplasty may be a new surgical approach with which to manage corneal endothelial disorders.

Bilateral, anterior stromal ring opacity of the cornea.

AIMS/BACKGROUND: To describe a bilateral, mid peripheral, ring-shaped corneal opacity, not resembling any known corneal degeneration, dystrophy, or other disorder, and occurring without ocular or systemic disease. METHODS: Ophthalmic examination, haematological screening, and ultrasound biomicroscopy. RESULTS: A 25 year old man showed grey-white, granular opacities in both corneas, with an 8 mm diameter ring configuration, and a V-shaped distribution in the anterior stroma. The surrounding corneal stroma was clear, and the tear film, the epithelium and its basement membrane, Descemet's membrane, and the endothelium were normal. Evidence of systemic disease was not found. Family members did not show corneal abnormalities. CONCLUSION: A bilateral corneal ring opacity may occur in healthy, asymptomatic, young people. These corneal rings may result from depositions of unknown origin, or possibly a rare corneal dystrophy.

A new surgical technique for deep stromal, anterior lamellar keratoplasty.

AIMS: To describe a new surgical technique for deep stromal anterior lamellar keratoplasty. METHODS: In eye bank eyes and sighted human eyes, aqueous was exchanged by air, to visualise the posterior corneal surface--that is, the "air to endothelium" interface. Through a 5.0 mm scleral incision, a deep stromal pocket was created across the cornea, using the air to endothelium interface as a reference plane for dissection depth. The pocket was filled with viscoelastic, and an anterior corneal lamella was excised. A full thickness donor button was sutured into the recipient bed after stripping its Descemet's membrane. RESULTS: In 25 consecutive human eye bank eyes, a 12% microperforation rate was found. Corneal dissection depth averaged 95.4% (SD 2.7%). Six patient eyes had uneventful surgeries; in a seventh eye, perforation of the lamellar bed occurred. All transplants cleared. Central pachymetry ranged from 0.62 to 0.73 mm. CONCLUSION: With this technique a deep stromal anterior lamellar keratoplasty can be performed with the donor to recipient interface just anterior to the posterior corneal surface. The technique has the advantage that the dissection can be completed in the event of inadvertent microperforation, or that the procedure can be aborted to perform a planned penetrating keratoplasty.

Scar tissue orientation in unsutured and sutured corneal wound healing.

AIMS: This study aimed to evaluate stromal wound healing morphology in short term unsutured compared with sutured corneal wounds, to define regional variation in healing within radial keratotomy wounds. METHODS: Stromal scar tissue orientation (fibroblast and collagen fibre orientation) was analysed in unsutured and adjacent sutured keratotomy wounds in monkeys, 2 to 9 weeks after surgery, using light and transmission electron microscopy. RESULTS: At 2 to 4 weeks, scar tissue orientation was transverse to the wound edge in unsutured wounds, but sagittal in sutured wounds. At 5 to 9 weeks, a reorientation of scar tissue sagittal to the wound was seen in the unsutured wounds, proceeding from the posterior to anterior wound regions. In sutured wounds, a scar tissue reorientation transverse to the wound was seen, proceeding from the anterior wound region in a posterior direction. CONCLUSIONS: Within the same cornea, sutured and unsutured wounds showed opposite patterns of healing. Sutured wounds initially healed more slowly, but obtained pseudolamellar continuity over time. In contrast, healing of unsutured wounds was characterised by an early approximation towards lamellar repair that was followed by an ineffective reorganisation of the scar. This latter pattern of healing, that may be associated with a variable weakening of the wound, may relate to the clinical findings of unpredictability and/or progression of refractive effect following radial keratotomy.

Double vital staining using trypan blue and infracyanine green in macular pucker surgery.

AIMS: To study the clinical properties of double vital staining in premacular fibrosis, facilitating complete removal of all epiretinal tissue. METHODS: In a two step surgery, the epiretinal pucker was removed after staining with trypan blue, whereafter the inner limiting membrane was peeled after staining with infracyanine green. RESULTS: In all 30 patients, a separate epiretinal layer and inner limiting membrane were removed from the macular area. Pathological examination showed different histological properties of the removed layers. An increased visual acuity was measured in 26 patients, and a slightly decreased visual acuity in one patient. CONCLUSION: The described double staining technique could be a novel valuable tool that may help to achieve optimal anatomical and functional recovery after surgery for premacular fibrosis

Healing of reopened-and-sutured radial keratotomy wounds.

We designed a study to evaluate healing in reopened-and-sutured (RAS) keratotomy wounds to determine the efficacy of reoperations in treating radial keratotomy overcorrections. Using light and transmission electron microscopy, we compared stromal scar tissue organization (transverse fibroblast orientation and collagen fiber continuity across the wound) in RAS wounds and in sutured and unsutured control wounds in 18 monkey eyes one to nine weeks after surgery. Wound healing morphology of RAS wounds varied with the interval between reoperation and termination of the experiment. Scar tissue organization was sagittal at one week postoperatively, transverse in the anterior and mid regions after four weeks, and transverse over the entire wound after nine weeks. Sutured wounds showed a similar pattern of healing, although transverse scar tissue organization was restricted to the anterior and mid regions in the late healing phases. In contrast, unsutured wounds showed a temporary, transverse scar tissue organization over the entire wound depth at two to four weeks and a progressive reorientation of the mid and posterior scar tissue sagittal to the wound at later intervals. The results suggest that reopening and suturing keratotomy incisions to treat radial keratotomy overcorrections may be effective through a myopic shift induced by sutured wound apposition and long-term wound remodeling, contraction, or both.

Three versus four radial keratotomy incisions.

Radial keratotomy (RK) is currently performed with four or eight semi-radial incisions. To evaluate the effect of a theoretically more stable three-incision RK pattern, centripetal incisions were made in 16 human donor eyes (eight pairs), using a double-edged diamond blade set to 90% of central pachymetry and a 3.5 mm optical clear zone. Intraocular pressure was maintained at 15 mm Hg during surgery and while keratometry readings were made. One randomly selected eye of each pair had three radial incisions made at 12, 4 and 8 o'clock; the other eye had four radial incisions at 12, 3, 6, and 9 o'clock. Corneal flattening was 6.08 diopters (D) with four incisions and 4.84 D with three incisions (P less than .05). Astigmatism increased 0.44 D and 0.69 D, respectively (P greater than .1). Histologically measured mean incision depth (77.4%) did not differ significantly between the groups (P greater than .1). This study shows that 80% of the effect of a four-incision RK pattern can be obtained with a theoretically more stable three-incision pattern.

Trypan blue capsule staining to visualize the capsulorhexis in cataract surgery.

A capsulorhexis may be difficult to perform in the absence of a red fundus reflex. Using 0.1 mL of trypan blue 0.1% to stain the anterior capsule in 30 patients with a mature cataract enabled us to visualize the capsulorhexis during phacoemulsification. No adverse reactions were observed up to 12 months after surgery. Trypan blue staining of the anterior capsule appears to be a safe technique to facilitate the performance of a capsulorhexis in the absence of a red fundus reflex.

A quick surgical technique for deep, anterior lamellar keratoplasty using visco-dissection.

PURPOSE: To describe a new surgical technique for deep, anterior lamellar keratoplasty using a viscoelastic for dissection of Descemet's membrane (DM) from the posterior stroma. METHODS: Through a paracentesis, aqueous was exchanged by air to visualize the posterior corneal surface-i.e., the air-to-endothelium interface. Using the interface as a reference plane, a 30 gauge needle was inserted into the cornea to just anterior to DM. Viscoelastic was injected to separate DM from the posterior stroma, and a recipient, anterior lamella was excised. A full-thickness donor button was sutured into the recipient bed, after stripping its DM. RESULTS: In 25 eye bank eyes, the procedure could be completed in 20 eyes; in 5 eyes, DM ruptured during visco-dissection. With light microscopy, dissection depth was located at the level of DM. In two patient eyes the procedure could be completed. In a third patient eye DM ruptured during visco-dissection, and the procedure was converted into a penetrating keratoplasty. CONCLUSION: Using visco-dissection, a lamellar keratoplasty can be performed quickly, with the donor-to-recipient interface just above the recipient DM, i.e., with a nearly perfect anatomical replacement of all corneal stroma. There is substantial risk of rupture or microperforation of DM during surgery.

Depth predictability of stromal pockets in the posterior cornea.

PURPOSE: To evaluate the predictability of the depth of stromal pockets made in the posterior cornea for the excision of anterior or posterior lamellar corneal buttons with a planned thickness. METHODS: Stromal corneal pocket dissections were created in human eye bank eyes by making a peripheral arcuate keratotomy incision at 60, 80, or 95% of central pachymetry and creating a pocket from the bottom of the incision across the cornea. Pocket depth was measured by pachymetry immediately after surgery and by light microscopy. RESULTS: Mean achieved central pocket depth differed by 0.03+/-0.03 mm from the intended depth. Variation in depth across the pocket decreased from 0.07+/-0.02 mm for pockets made at 60% of the intended depth to 0.05+/-0.01 mm for pockets made at 80% depth, and 0.04+/-0.02 mm for pockets made at 95% depth (p < 0.01). Pachymetric and histological measurements of relative pocket depth averaged 64+/-9% and 73+/-7%, respectively, for pockets made at 60% of the intended depth, 82+/-7% and 86+/-3% for pockets made at 80% depth, and 91+/-7% and 92+/-3% for pockets made at 95% depth. The difference between pachymetric and histological relative pocket depth measurements decreased with deeper pocket depth (p < 0.01). CONCLUSIONS: In the posterior cornea, stromal pockets can be created to within 30 microm from the intended depth. Variation in depth throughout the pocket decreases with deeper pocket depth. Pachymetry is a reliable method to check the achieved pocket depth during surgery; the accuracy of pachymetry readings improves with deeper pocket depth.

A technique to visualize corneal incision and lamellar dissection depth during surgery.

PURPOSE: To describe a surgical technique to visualize the depth of corneal incisions and lamellar stromal dissections during surgery. METHODS: In porcine cadaver eyes, the aqueous was exchanged by air. Thus an air-to-endothelium interface (i.e., a useful optical surface) was created at the posterior corneal surface. The air-to-endothelium interface was used as a reference plane to visualize the corneal thickness and the relative depth of corneal incisions and dissections. Freehand peripheral corneal incisions, tangential keratotomy incisions, and lamellar stromal dissections were made at an intended corneal depth of 60, 80, and 99%. Light microscopy was used to measure the relative depth of the incisions and dissections. RESULTS: Achieved depth for peripheral corneal incisions averaged 65.2+/-5.3%, 78.8+/-5.1%, and 93.4+/-6.0%, respectively (p<0.05); and for tangential keratotomy incisions, 68.2+/-7.3%, 83.2+/-4.4%, and 95.8+/-3.6%, respectively (p<0.05). Achieved depth for lamellar stromal dissections averaged 58.3+/-9.4%, 81.1+/-3.4%, and 94.4+/-1.5%, respectively (p<0.05). Microperforations occurred with three incisions made at 99% intended depth. CONCLUSION: During surgery, the depth of incisions and lamellar dissections relative to the corneal thickness can be visualized by filling the anterior chamber with air (i.e., by creating an optical interface at the posterior corneal surface).

A surgical technique for posterior lamellar keratoplasty.

PURPOSE: To design a surgical technique for transplantation of posterior corneal tissue, while leaving the recipient anterior cornea intact. METHODS: In human cadaver eyes, and in a cat and monkey model, recipient eyes had an 8.0-mm limbal incision made with a diamond blade set to 50% of central pachymetry. A stromal pocket was created across the cornea, and a 6.0-mm diameter posterior lamellar disc was excised. A donor posterior disc was implanted into the recipient opening, and the limbal incision was sutured. The procedure was evaluated with keratometry, biomicroscopy, endothelial (supra)vital staining, and light microscopy. RESULTS: In human cadaver eyes, post-operative astigmatism averaged 1.2 D (SD, +/- 0.6 D). Posterior transplants showed an intact endothelial cell layer with 1.0% (SD, +/- 1.2%) of cell death. In the animals, six (75%) eyes had clear transplants 2 weeks after surgery; one of these eyes later developed an allograft rejection. Two (25%) eyes showed corneal decompensation, because of inverted implantation of the donor disc. Microscopy showed minimal scarring at the donor-to-host interface and a normal wound-healing response at the posterior stromal wound edges. CONCLUSION: In experimental models, posterior lamellar keratoplasty can be performed through a limbal incision and a mid-stromal pocket. The procedure may be a potential alternative in the surgical management of corneal endothelial disorders.