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Doi:10.1016/j.ophtha.2006.01.07

Deep Lamellar Keratoplasty
by Intracorneal Dissection
A Prospective Clinical and Confocal Microscopic Study Giorgio Marchini, MD,1 Leonardo Mastropasqua, MD,2 Emilio Pedrotti, MD,1 Mario Nubile, MD,2Marco Ciancaglini, MD,2 Arianna Sbabo, MD1 To evaluate the clinical findings, visual outcomes, and confocal microscopic corneal features of a surgical technique for manual deep lamellar keratoplasty (DLKP) with intentional sparing of the most posterior stroma.
Noncomparative, prospective, 12-month interventional study.
Forty-six eyes of 45 patients who had corneal pathologic features without endothelial abnor- malities and requiring corneal graft were treated by DLKP by manual stromal delamination. They were examinedclinically after surgery and using in vivo confocal microscopy at 2 weeks and 1, 3, 6, and 12 months.
The surgical technique consisted of an intracorneal deep manual stromal dissection through a 4-mm limbal incision at 50 ␮m from Descemet's membrane (DM). After trephination, an endothelial free graftwas sutured.
Main Outcome Measures:
Topographic parameters, interface depth and reflectivity, and anterior and postinterface keratocyte density; visual acuity was correlated with these parameters.
Two eyes had rupture of the DM. Two eyes that had delayed epithelial healing because of graft override with stromal inflammation underwent a second surgery (penetrating keratoplasty). Mean uncorrectedlogarithm of the minimum angle of resolution (logMAR) uncorrected visual acuity and logMAR best-correctedvisual acuity (BCVA) improved from preoperative values (1.342⫾0.239 and 0.923⫾0.226, respectively) to0.421⫾0.122 and 0.104⫾0.068, respectively, at 12 months. Mean topographic astigmatism was 3.09⫾1.30diopters (D) at 3 months after suture adjustment, and 2.87⫾0.92 D at 12 months after suture removal. Averageinterface depth was 64.2⫾6.7 ␮m at 15 days and showed no significant changes up to 12 months. Meaninterface reflectivity was highest at 15 days (95.5⫾15.7 light reflectance units [LRU]) and showed a progressivedecrease over time of 55.3⫾8.7 LRU at 12 months. A significant negative correlation was observed betweenBCVA and topographic astigmatism up to 1 month and between BCVA and interface reflectivity starting from 6months after surgery.
Deep lamellar keratoplasty by intracorneal dissection provides visual and clinical results comparable with that of other DLKP techniques. Visual recovery is slow and progressive, taking up to 1 year.
Confocal microscopy enables precise evaluation of corneal features, interface morphologic features, andreflectivity, demonstrating a negative correlation between interface reflectivity and BCVA showing that theprogressive recovery over months of the interface transparency is correlated with the increase in visual acuityafter 6 months. Ophthalmology 2006;113:1289 –1300 2006 by the American Academy of Ophthalmology. Lamellar keratoplasty (LKP) is a corneal graft technique able aspects with respect to penetrating keratoplasty (PKP).
consisting in the transplantation of partial thickness donor Because it is an extraocular procedure, it has a more limited cornea without endothelium, Descemet's membrane (DM), risk of complications, such as anterior synechia, secondary and rear stroma onto a recipient healthy stromal bed after glaucoma, or Because LKP maintains the dissection of pathologic anterior cornea. It presents favor- recipient's endothelium in place, there is no risk of endo-thelial rejection and consequently no need for long-termimmunosuppressive therapy, decreasing the incidence of Originally received: July 7, 2005.
infection, glaucoma, and It also offers a superior Accepted: January 24, 2006.
Manuscript no. 2005-621.
wound strength, the sutures can be removed earlier, and it 1 Eye Clinic, Department of Neurological and Visual Sciences, University requires less rigid criteria for donor corneal tissue selec- of Verona, Verona, Italy.
Despite these advantages, the main clinical concern 2 Department of Medicine and Aging Science, Ophthalmic Clinic, Univer- that limits the applications of LKP as a therapeutic kerato- sity of Chieti-Pescara, Chieti-Pescara, Italy.
plasty is that the resulting visual acuity is not as good as that Correspondence to Emilio Pedrotti, MD, Eye Clinic, Department of Neu- obtained with The reason for the poor visual rological and Visual Sciences, University of Verona, Borgo Trento Hos-pital, Piazzale Stefani, 1, Verona 37126, Italy.
acuity after LKP is thought to be the scarring that involves 2006 by the American Academy of Ophthalmology ISSN 0161-6420/06/$–see front matter Published by Elsevier Inc.
Volume 113, Number 8, August 2006 the interface between the graft cornea and the recipient point of more than 300 ␮m; and (4) absence of other ocular stromal bed. Deep lamellar keratoplasty (DLKP) has been pathologic features such as glaucoma or retinal diseases. The proposed as a surgical alternative procedure to avoid inter- original diseases were keratoconus with contact lenses intolerance face It consists of the removal of all recipient (34 eyes), stromal dystrophies (5 eyes), corneal leucoma (5 eyes),and stromal opacity occurring after herpes keratitis (2 eyes). All stromal tissue down to DM and the transplantation of the patients enrolled in the study gave informed consent, and the donor cornea without endothelium over the host bed. Deep principles of the Helsinki Declaration and good clinical practice lamellar keratoplasty presents the advantage of a greater were followed. A complete ocular examination, including uncor- improvement in postoperative visual acuity with respect to rected visual acuity (UCVA) and best-corrected visual acuity LKP and compares favorably with PKP while maintaining (BCVA) evaluation, slit-lamp biomicroscopy, applanation tonom- the same advantages of Deep lamellar kerato- etry, fundus examination, computerized topography, specular mi- plasty is indicated in many pathologic features where PKP croscopy endothelial cell density, and ultrasound pachymetry were was previously regarded as the first surgical choice in pa- performed before surgery. Each patient was examined in the post- tients with healthy endothelium and the absence of stromal operative period at 15 days and 1, 3, 6, and 12 months after edema, such as corneal scarring resulting from keratitis, surgery. Each follow-up visit included UCVA and BCVA, slit-lamp biomicroscopy, endothelial specular microscopy (Noncon corneal dystrophies, and The main concern Robo-P, Konan, Hyogo, Japan), ultrasound pachymetry (Ophtha- regarding DLKP is the difficulty of the surgical procedure: sonic, Teknar, St. Louis, MO), and in vivo confocal microscopy of perforation of DM is the main intraoperative risk, especially the cornea (Confoscan 3, Nidek Technologies, Padova, Italy).
when DM is completely exposed free of overlying If Visual acuity measurements were performed using Snellen acuities perforation occurs, even if DLKP can be completed in some and were analyzed using logarithm of the minimum angle of cases, the stromal dissection may be difficult, leading to a resolution (logMAR) equivalents for statistical analysis. Corneal low-quality corneal bed. Furthermore, when conversion of the topography was performed using an EyeTop instrument (CSO procedure into PKP is needed because of significant breaks in Ophthalmic, Firenze, Italy), and topographic astigmatism was DM, good quality endothelium of donor cornea may not be measured using a simulated keratometry value given by the power It is not clear whether a complete stromal excision and axis of the steepest meridian and that of 90°. Surface regularityindex (SRI) and surface asymmetry index (SAI) were analyzed to baring DM provides better visual results than cases in which a study quantitatively the irregularity of topographic small portion of rear stroma is left undissected. Sugita and Intraoperative and postoperative complications also were recorded.
reported that leaving a small amount of stroma doesnot produce differences in visual acuity compared with com- plete stromal dissection, as long as the deep stroma is healthy.
Thus, intentional sparing of the deepest stroma could be help- All the DLKP surgeries were performed under general anesthesia ful in preventing intraoperative DM rupture.
at the Eye Clinic of the University of Verona. To facilitate and We developed a new kit of instruments for DLKP based on speed up the procedure, we designed a special surgery kit com- blunt spatula manual delamination, and we named the surgical posed of a precalibrated diamond knife (Janach, Como, Italy), a technique DLKP by intracorneal dissection; this technique 2.25-mm surgical knife with a round head angled 60° with respectto the handle (BD Edge Ahead Circular knife, BD Ophthalmic intentionally leaves a minimal thickness of deep stroma on the Systems, Bidford-on-Avon, United Kingdom), a double-ended host bed, without the aid of air or fluid in dissection, before spatula (short and long) for stromal tissue delamination (Janach, suturing the donor button. The theoretical advantages of such Como, Italy), a Hanna trephine (Moria, Paris, France), forceps for a technique are: (1) the reduced risk of intraoperative compli- stromal peeling and for donor button endothelium removal (Ja- cations such as DM rupture and the formation of a double nach), and a punch for donor button (Moria). Central and periph- anterior chamber, and (2) the lower complexity of the DLKP eric corneal thicknesses were measured using an ultrasonic procedure, generally demanding greater surgical skills to han- pachymeter (Ophthasonic, Teknar, St. Louis, MO). The surgical dle correctly the extremely thin DM– endothelium layer.
procedure started with a 4-mm limbal incision with a precalibrated The purpose of this prospective study was to evaluate at diamond knife at the 10-o'clock position. The knife calibration 12 months of follow-up the complication rate, visual out- was set to the value of the thinnest corneal thickness minus 50 ␮mto spare the endothelium, DM, and a thin deep stromal layer. Then come, confocal microscopic interface features, and their a cleavage plane was formed by creating a small stromal pocket correlation with visual acuity in a cohort of patients treated with the angulated bevel up. Subsequently, this pocket was en- with DLKP by manual intracorneal dissection.
larged with the short dissector starting the deep stroma dissection.
The intrastromal cleavage delamination continued with the longdissector to dissect 80% of the stromal extension, or at least Patients and Methods
extending across an area that covered the central 9-mm diameter.
The deep stromal pocket was filled up with low cohesive and low Patient Population and Clinical Examinations
molecular weight viscoelastic (IAL-F, Fidia Farmaceutici S.p.A.,Abano Terme, Italy). This kind of viscoelastic was chosen to Between August 2001, and July 2003, 46 eyes of 45 patients (29 reduce the risk of endothelium perforation during the trephination males and 17 females; mean age, 35.6⫾14.1 years; range, 6 –70 of recipient button with Hanna trephine. The recipient button was years) were enrolled consecutively in this prospective noncom- 7.75 mm in 7 cases, 8.00 mm in 31 cases, and 8.25 mm in 8 cases.
parative study. All eyes underwent deep lamellar keratoplasty A sclerocorneal button stored in organ culture at 31° C and (DLKP) with intracorneal dissection technique. Criteria for eligi- provided by the Eye Bank Fondazione Banca degli Occhi del bility were: (1) corneal pathologic features requiring graft without Veneto (Venezia-Mestre, Italy) was used for the preparation of the evidence of endothelial impairment; (2) endothelial cell density donor cornea in all cases. The donor cornea was trephined from the more than 1800 cell/mm2; and (3) corneal thickness at the thinnest endothelial side with Moria punch with a 0.25-mm bigger diameter Marchini et al 䡠 Deep Lamellar Keratoplasty by Intracorneal Dissection than the recipient bed. Endothelium and DM were removed from cluded in the scan. Because the reliability of the Z-scan graphic is the donor button with dedicated forceps. This was carried out by influenced by eye movements during the scan period, only reliable placing the donor button on a sterile plastic support with the and comparable graphics for each corneal examination were con- endothelial face up. While a Pierce forceps blocked the button, a sidered for analysis.
second dedicated forceps peeled out DM and endothelium. Four Interface was defined as the corneal sublayer located in the interrupted 10-0 nylon sutures (Ethilon Black, Johnson & Johnson, posterior stroma with evident discontinuity of the stromal kerato- St. Stevens Woluwe, Belgium) at 12, 6, 3, and 9 o'clock secured cyte and extracellular matrix architecture. The interface depth was the corneal button in the recipient bed. Then, a single continuous calculated as the distance between the proper interface and the nylon 10-0 running suture (NU-1, Alcon Laboratories, Inc., Fort endothelial layer. Each value of interface depth was considered as Worth, TX) with 20 bites was placed. After removal of the inter- the average of 3 measurements obtained from 3 reliable Z-scan rupted suture, intraoperative adjustment of astigmatism was per- graphics. Interface reflectivity also was calculated as the average formed using the Maloney keratometer (Duckworth and Kent, of 3 light reflectance unit values obtained by 3 reliable Z-scan Baldock, UK). An association of 0.05% formocortal and 0.03% sulphate gentamicin was instilled topically at the end of the sur- Mean keratocyte density of anterior and postinterface residual gery. An extended-wear therapeutic soft contact lens (Protek T&S, stroma was calculated using the analysis software provided with Contact Vision, Marcon, Italy) was placed on the cornea and the the instrument, which allows manual cell count within a selected eye was patched.
region of interest of standardized dimension (0.1 mm2). Keratocyte Postoperative therapy included the topical application 6 cell density values of anterior (depth range, 50 –100 ␮m from the times daily of a corticosteroid-antibiotic solution composed of basal lamina of epithelium) and residual host stroma (between 0.1% bisodium phosphated betamethasone, 0.4% chloramphen- endothelium and interface layers) were obtained as an average of icol, 0.421% tetracycline, and 18 000 000 IU of sodic colis- 5 images per eye.
timethate. Eye drops were tapered within 3 months after surgery Confocal microscopy was performed before surgery in all eyes with the following schedule: 6 times daily for 20 days, 5 times to assess endothelial cell density. Postoperative endothelial cell daily for 10 days, 4 times daily for 10 days, 3 times daily for 10 count was obtained for each eye at every follow-up examination.
days, 2 times daily for 10 days, once daily for 10 days, and on The endothelial layer was imaged correctly for all patients. The alternate days for the final 20 days.
mean of 3 measurements of endothelial cell density was calculated Suture adjustments were performed when topographic astigma- using Confoscan 3 dedicated analysis software and performing a tism was superior to 3.5 diopters (D) within the third and fourth manual cell count processing within a 0.1-mm2 standardized re- postoperative week. In case of regression of the effect, postoper- gion of interest in the central cornea.
ative adjustment was repeated for a maximum of 3 times up to 3months after surgery. Suture removal was performed 6 to 8 months after surgery.
Statistical analysis was performed with SPSS 10.1 for Windows(SPSS Inc., Chicago, IL). Data were expressed as mean⫾standard In Vivo Confocal Microscopy
deviation, and a P value of less than 0.05 was considered statisti- In vivo confocal microscopy (IVCM) was performed using a cally significant. Interface depth, interface reflectivity, keratocyte scanning slit confocal microscope (Confoscan 3). This instrument density values, topographic astigmatism, SRI and SAI values, and was equipped with an Achroplan (Zeiss, Oberkochen, Germany) visual parameters (UCVA, BCVA) at different follow-up exami- nonapplanating ⫻40 immersion objective lens designed for full- nations were compared using a 1-way analysis of variance.
thickness examination of the cornea, with a working distance of Tukey's post hoc test was used to detect statistically significant 1.92 mm and a motorized focusing device. The center of the differences between values. The correlation between postoperative cornea was studied during all examinations. A drop of topical visual parameters and interface depth, interface reflectivity, ker- anesthetic 0.4% oxybuprocaine chlorohydrate (Novesina, Novartis atocyte density, and topographic parameters was analyzed using Farma S.p.A., Origgio, Italy) was instilled before the examination.
The patient was seated in front of the microscope, a chin rest anda forehead support were used, and the patient was asked to fix a bright object with the contralateral eye to minimize eye movementduring the examination. One drop of 0.2% polyacrylic gel (Vis- Graft Clarity and Complications
cotirs Gel, CIBA Vision Ophthalmics, Marcon, Venezia, Italy)was applied on the objective tip to serve as an immersion fluid.
The surgical procedure was completed in 45 of 46 eyes, and 2 Two complete confocal examinations of the entire central cor- intraoperative complications were observed. In 1 eye, a microper- nea were performed for each eye with a total examination time of foration of DM occurred during the trephination phase, but this did less than 5 minutes. The ConfoScan 3 acquires 350 images per not affect the subsequent surgical steps, leading to the uneventful examination at a rate of 25 frames per second, and therefore, the completion of the DLKP procedure. This was because the perfo- capture time is 14 seconds. The images are stored in memory and ration was less than 2 mm and out of the optical zone and because directly saved to the hard drive. Each frame is approximately the anterior chamber was maintained by an injection of a high 400⫻300 ␮m, with a scanning area of 0.12 mm2. The main molecular weight viscoelastic. One eye had a bigger perforation parameters for the sequence acquisition were set using 3 passes, during stromal dissection phase requiring conversion of the pro- for each complete corneal examination and a z-axis movement cedure into PKP. In this case, the DM– endothelium rupture was on range of 1000 ␮m, giving a theoretical z-axis distance between the optical zone, and the viscoelastic was unable to maintain the images in the scans of 10 ␮m. The position on the z-axis of the anterior chamber. A full-thickness corneal trephination similar corneal thickness of each image was obtained using the Z-scan to a standard PKP, followed by the apposition of an entire function of the instrument. The Z-scan is a graph showing the corneal button sutured with a single continuous running suture, depth coordinate (expressed in micrometers) on the z-axis and the was performed.
level of reflectivity (expressed in arbitrary numerical units, called Delayed epithelial healing resulting from graft override with light reflectance units) on the y-axis for each corneal image in- stromal inflammation, unresponsive to therapy, occurred within 3 Volume 113, Number 8, August 2006 Figure 1. Graph showing the mean postoperative logarithm of the minimum angle of resolution (logMAR) uncorrected visual acuity (UCVA) and
best-corrected visual acuity (BCVA) changes over time after deep lamellar keratoplasty. Each increment of both UCVA and BCVA was significant with
respect to previous time values. Note that the greatest increase of BCVA was between 3 and 6 months, whereas topographical parameters did not show
significant changes at this time point.
months after surgery in 2 eyes. Both eyes were treated with a provement of both BCVA and UCVA over time, at each follow-up subsequent PKP. A mild button haziness and edema were recorded examination, was observed. Notable relationships between speed in 1 eye 7 months after DLKP. This complication underwent of recovery of visual acuity and either age or original disease were complete resolution and normal button transparency was achieved not observed.
within 7 days of medical treatment (dexamethasone 0.2% eye- Mean postoperative corneal topographic astigmatism was drops, 6 times daily for the first week and then tapered during the 6.86⫾3.51 D 15 days after surgery, 3.09⫾1.30 D at 3 months after following 8 weeks). No other complications occurred in any eyes eventual suture adjustment, and 2.87⫾0.92 D at 12 months after over the entire follow-up period.
suture removal. The mean time of suture removal after surgery was With respect to graft clarity, an interface mild opacity, visible 209⫾62 days. Variations of mean corneal topographic astigma- at slit-lamp examination, was noted in all cases during the first tism, SRI, and SAI over the 12-month follow-up are reported in period after surgery. This slight interface haziness presented Ninety-one percent of eyes (39 of 43) underwent 1 or biomicroscopic clinical characteristics of edema that resolved more (up to 3) suture adjustments between 15 days and 3 months within 2 to 4 months after DLKP. However, the donor corneal after surgery. Topographic data at 15 days indicated postoperative button was found to be biomicroscopically clear in all eyes during astigmatism before suture adjustment and at 1, 3, and 6 months the entire follow-up, except in those 2 patients who experienced after eventual suture adjustments, and at 12 months after suture the above-mentioned postoperative complications. Epithelial removal. A significant overall decrease (P⬍0.001, analysis of pathologic features, such as epithelial edema or microerosions, variance) of topographic astigmatism was related to the differences were not observed in any eye.
between 0.5 to 1 month and 1 to 3 months Surfaceregularity index and SAI postoperative values showed a significanttendency toward decrease (P ⫽ 0.013 and P ⫽ 0.004, respectively, Visual Outcome and Topographic Astigmatism
analysis of variance). However, the comparison at different time In the 43 eyes observed for 12 months, the average preoperative points indicated a significant difference only for SRI between 0.5 logMAR BCVA was 0.923⫾0.226, and the mean postoperative and 12 months and for SAI between 0.5 and 6 to 12 months, as logMAR BCVA improved to 0.104⫾0.068 at the end of follow-up (P⬍0.001). Average logMAR UCVA improved from a preopera-tive value of 1.342⫾0.239 to 0.421⫾0.122 at 12 months after In Vivo Confocal Microscopy
surgery (P⬍0.001). In 41 of 43 eyes (95.3%), logMAR BCVA was0.2 or better, whereas in 30 of 43 eyes (69.7%), it was 0.1 or better, A deep lamellar interface was identified in all examined eyes at all at 12 months. shows the changes in postoperative BCVA follow-up examinations. This appeared at confocal examination as and UCVA during the follow-up. A statistically significant im- a corneal sublayer with unique morphologic characteristics in the

Marchini et al 䡠 Deep Lamellar Keratoplasty by Intracorneal Dissection Table 1. Topographic Astigmatism, Surface Regularity Index, face morphologic features at different postoperative times are and Surface Asymmetry Index during Follow-up presented in a typical Z-scan graphic showing the inter-face depth determination is presented in Mean interface depth (defined as distance from endothelium) at 15 days after surgery was 62.2⫾6.7 ␮m (range, 45–74 ␮m) and remained stable without significant variations during the entire follow-up Mean interface reflectivity values at each examination are reported in Interface reflectivity was found to be highest at 15 days and at 1 month (95.5⫾15.7 and 84.5⫾8.4 light reflectance units, respectively) and presented asubsequent and progressive decrease, indicating that tissue trans- D ⫽ diopters.
parency was regained. Statistically significant changes were ob- *P⬍0.001 between 0.5 and 1 month.
served between 15 days and 1 month and between 3 and 6 months.
†P⬍0.001 between 1 and 3 months.
Morphologically, the cause of the increased reflectivity of the P ⫽ 0.007 between 0.5 and 12 months.
interface, evidenced in the first period after surgery, was likely to be P ⫽ 0.020 between 0.5 and 6 months.
储P ⫽ 0.003 between 0.5 and 12 months (analysis of variance–Tukey post interface edema presenting an accumulation of reflective interface fluid, rather than cellular activation or interface scarring Mean anterior and postinterface stromal keratocyte density values at every examination are shown in No statistically cornea represented by evident discontinuity of stromal cellular and significant changes in anterior and posterior mean cell density extracellular architecture of the overlying and underlying rear were observed during the follow-up period. Eyes with keratoco- stroma, absence of distinguishable keratocytes, presence of homo- nus, but not those affected by other diseases, showed that the geneous reflectivity, variable transparency, and possible presence stromal layers underlying the interface (i.e., host residual stromal of bright microdots. In vivo confocal microscopy images of inter- tissue) presented hyporeflexive streaks. shows a sequence Figure 2. Confocal images of the interface of the same patient at (A) 15 days, (B) 3 months, (C) 6 months, and (D) 12 months after deep lamellar
keratoplasty. Note the progressive reduction of haziness and brightness, associated with the recovery of transparency: keratocytes became visible from
adjacent layers, as well as some bright microinclusions similar to those visible in LASIK interfaces.
Volume 113, Number 8, August 2006 Figure 3. Z-scan graphic showing interface depth determination: 3 consecutive Z-scan graphics of a confocal scan 15 days after surgery. Endothelial and
epithelial reflectivity peaks were visible in the left and right edge of each graphic, respectively. A preendothelial high reflective peak representing the
interface reflectivity spike was visible (arrow). The green band on the second curve represents the z-axis distance between the interface and the endothelial
layer.
of images of the posterior corneal layers, including interface, after BCVA and topographic astigmatism, SRI, and SAI up to 1 month DLKP for keratoconus.
after surgery. Best-corrected visual acuity and interface reflectivity Endothelial cell density values recorded over the follow-up are values showed a significant negative correlation starting from 6 shown in Mean preoperative endothelial cell density was months Other tested parameters did not show a signif- 2565⫾512 cell/mm2, whereas it was 2210⫾489 cell/mm2 at 15 icant correlation with visual acuity at the follow-up time points.
days after surgery (cell loss with respect to preoperative values,13.8%; P⬍0.001) and 2135⫾501 cell/mm2 at 1 month (cell losswith respect to 15-day values, 3.4%; P ⫽ 0.048). Ensuing changes that were detected were not significant.
Several authors have reported a favorable visual outcome after Recent results of comparison studiessuggest that DKLP can be considered as a safe alternative to Uncorrected visual acuity showed a significant negative correla-tion with topographic astigmatism during the entire follow-up. A PKP in the treatment of different anterior corneal pathologic significant negative correlation between UCVA, SRI, and SAI characteristics with stromal opacity and in keratoco- were detected only up to 1 month and 3 months, respectively In addition to comparable results in terms of A significant negative correlation was observed between visual recovery, DLKP presents several advantages with Table 2. Interface Reflectivity, Interface Depth, and Mean Anterior and Posterior Keratocyte Density Changes over Follow-up Time (mos)
Statistically significant changes were observed for interface reflectivity only between 0.5 and 1 month and 3 to 6 months after surgery.
Postoperative endothelial cell density showed a slightly significant decline between 0.5 and 1 month. A greater endothelial cell loss was observed betweenpreoperative and 0.5-month postoperative values (see text).
*P⬍0.001 between 0.5 and 1 month (Tukey post hoc test).
†P⬍0.001 between 3 and 6 months (Tukey post hoc test).
‡P ⫽ 0.048 between 0.5 and 1 month (Tukey post hoc test).

Marchini et al 䡠 Deep Lamellar Keratoplasty by Intracorneal Dissection Figure 4. Sequential confocal scan images of the central cornea 12 months after deep lamellar keratoplasty for keratoconus. The images represent different
corneal layers on the z-axis starting from the endothelial plane toward the posterior stroma (from top left to bottom right). The endothelial layer was
evident in the sequence (first line) on top (⬎1 endothelial image was captured). The posterior residual host stromal bed was clearly imaged in the following
frames, and typical stromal keratoconus hyporeflexive striae were visible within keratocytes and extracellular architecture (arrowhead). Preinterface and
postinterface stromal layers were imaged in the following frames of third and fourth lines. The proper interface (arrow) presented absence of distinguishable
keratocytes and presence of bright microinclusions that extend toward the layer adjacent to the interface. Note that the interface was optically clear with
low reflectivity. The last 2 frames of the fourth line indicated that the initial posterior stromal layers of donor button evidenced normal architecture of
keratocytes and extracellular tissue transparency (star).
respect to PKP, mainly linked to the preservation of corneal well-trained in corneal grafts. It is still an evolving technique, host endothelium, leading to a lower risk of endothelial lacks a standardization of procedures in the different indicating rejection and late endothelial as well as a greater pathologic characteristics, lacks long-term (years) results, and availability of donor corneas that do not need perfectly presents potential interface haze or irregularity, with the ensu- healthy endothelium and high endothelial cell density to be ing related negative influences on visual Inter- suitable for corneal grafting. Other favorable aspects con- face scarring leading to poor visual acuity represents one of cerning DLKP are represented by a faster stabilization of the greatest reservations that has limited the applications of the wound, leading to earlier suture and a lower LKP in the The deeper interface obtained risk of open sky surgery-related complications such as en- with new DLKP techniques has not been associated with dophthalmitis. However, different theoretical disadvantages a significant incidence of opacification and irregularity are thought to influence DLKP procedures. Deep lamellar occurring at the level of donor– host corneal lamellar keratoplasty is technically difficult, demanding a surgeon This aspect may affect the better visual Volume 113, Number 8, August 2006 Table 3. Pearson's Correlation between Logarithm of the Minimum Angle of Resolution Uncorrected and Best-Corrected Visual Acuity with Parameters That Showed Significant Correlation at a Minimum of 1 Follow-up Visit Time (mos)
r ⫽ ⫺0.700 r ⫽ ⫺0.780 r ⫽ ⫺0.647 r ⫽ ⫺0.513 r ⫽ ⫺0.695 r ⫽ ⫺0.491 r ⫽ ⫺0.370 r ⫽ ⫺0.21 r ⫽ ⫺0.253 r ⫽ ⫺0.218 P ⫽ 0.011 r ⫽ ⫺0.308 r ⫽ ⫺0.491 r ⫽ ⫺0.345 r ⫽ ⫺0.268 r ⫽ ⫺0.207 P ⫽ 0.037 P ⫽ 0.001 P ⫽ 0.019 r ⫽ ⫺0.695 r ⫽ ⫺0.352 r ⫽ 0.270 r ⫽ 0.220 r ⫽ 0.218 r ⫽ ⫺0.561 r ⫽ ⫺0.540 r ⫽ ⫺0.235 r ⫽ ⫺0.198 r ⫽ ⫺0.203 r ⫽ ⫺0.602 r ⫽ ⫺0.405 r ⫽ ⫺0.222 r ⫽ ⫺0.232 r ⫽ ⫺0.212 P ⫽ 0.009 r ⫽ ⫺0.104 r ⫽ ⫺0.073 r ⫽ ⫺0.273 r ⫽ ⫺0.734 r ⫽ ⫺0.945 P ⫽ 0.047 BCVA ⫽ best corrected visual acuity; NS ⫽ not significant; SAI ⫽ surface asymmetry index; SRI ⫽ surface regularity index; UCVA ⫽ uncorrected visualacuity.
acuity results of DLKP, which presents values comparable On the basis of the above-mentioned observations, we with those after performed a standardized surgical technique for lamellar A possible explanation for the better interface transpar- dissection with the aim of obtaining a reproducible DLKP, ency of DLKP over traditional LKP may be the smooth host using spatula-mediated manual deep delamination by which surface obtained by separating DM from the deepest stroma an intended host stromal residual layer of approximately 50 and the very slight optical disturbance of the donor stromal ␮m is left undissected above the DM–endothelial layer.
However, it has not been established clearly in When performing a dissection in a nontrephined cornea, it is literature which reasons are responsible for the different easier to extend the cleavage plane on the entire corneal area behavior affecting interface transparency of corneal tissues creating a wide pocket, which allows a better apposition of occurring either after LKP or DLKP. A recent report doc- the donor button on the recipient bed even in presence of umented that separation may occur between the anterior more advanced ectasic pathologic features.
banded and the posterior nonbanded layer of DM in some In the present study, we prospectively evaluated visual cases during DLKP.Several techniques of stromal dissec- results, complications rate and interface morphology, over a tion down to DM have been proposed over the past years to 12-month follow-up period in eyes that underwent DLKP facilitate a safe and efficient deep stromal by intracorneal dissection for keratoconus (34 eyes) or The original article by followed by other stud- anterior stromal opacity (12 eyes). The main outcome mea- described air injection into the stromal bed. Other sures chosen for this study were specifically related to the proposed delamination methods include hydrodelamination, parameters for evaluating donor– host interface characteris- in which a saline solution is used as a dissecting tics of the graft. The potential for interface haze, although viscoelastic dissection alone or after air injection into the minimal, is considered to be a possible occurrence interfer- anterior and the recent big bubble technique ing negatively with visual performance in any kind of based on air injection into the paracentral corneal stroma corneal lamellar grafts; therefore, an objective method for after partial-thickness Although providing interface evaluation seemed to us as to be an important issue smoother surfaces, baring the DM as a surgical goal requires for at least 2 reasons. First, although a recent study of DLKP that the surgeon deals with a very thin layer of residual host reported interface opacification as a relatively infrequent cornea (less than 40 ␮m), resulting in a variable incidence complication (incidence of 8% in the reported 25-patient (between 0% and 39%) of DM Sugita to our knowledge, quantitative methods have not and Kondo, when comparing patients in whom the DM was been used to measure interface transparency, and this has exposed with patients who retained some portion of non- been evaluated by using subjective slit-lamp biomicroscopic pathologic deep stroma left during surgery, reported that no examination. In fact, interface haziness should not be con- differences in visual acuity and endothelial cell density were sidered as a discrete parameter (i.e., present or not present), found in a prospective study during a 12-month but as a continuous variable inducing optical disturbance, It is possible, although not clearly proven in literature, that which may interfere differently with visual performance intentional sparing from dissection of a thin layer of healthy with a dependence on opacity location, quantity, and homo- rear stroma during deep lamellar grafts may provide the geneity. Moreover, the visual acuity obtained in previous advantage of reducing the risk of DM puncture, allowing studies of DLKP was correlated with interface transparency, surgeons to perform the delicate phase of deep delamination which may be a factor influencing visual acuity over time.
in a simpler manner.
Second, DLKP using deep delamination but without baring Marchini et al 䡠 Deep Lamellar Keratoplasty by Intracorneal Dissection DM theoretically may present greater problems of interface (DM exposed or some deep stromal layers left undissected optical quality (being somewhat similar in principle of above the endothelium). Moreover, Shimazaki et in their interface haze generation to LKP) than DLKP with DM DLKP series for corneal pathologic features, except from keratoconus, with 24-month follow-up observed that visual In vivo confocal microscopy has been used widely in acuity stabilized within 6 months after surgery and that no recent years as a valid diagnostic technique for the in vivo significant differences were observed during follow-up be- microscopic imaging of corneal structure, both in healthy tween these values and PKP group visual acuity score.
and pathologic One of the fields of application in However, in our patients, visual recovery was slow and which IVCM was shown to be a potentially useful tool is progressive, with continuous increments over time, as that of corneal refractive In vivo confocal mi- croscopy has been used to evaluate corneal structure and The results of this study thus indicate that visual acuity morphologic features in LASIK-treated Spe- outcomes, obtained with intentional sparing of the most cifically, IVCM has been shown to be effective in the posterior stromal layers, are comparable with other DLKP microscopic imaging of the LASIK flap–stroma interface, techniques baring the DM, confirming the observation by allowing identification of the interface layer, which is char- Sugita and but that visual recovery requires at least acterized by the presence of microscopic interface bright 6 to 12 months to reach satisfactory levels. Over the 12- variable reflectivity (low to month follow-up, we evaluated topographical parameters and disappearance of keratocytes around the wound inter- and interface microscopic morphologic parameters that In addition, IVCM allowed flap thickness and could affect visual performance over time.
residual stromal thickness measurements by using confocal Although starting from mean postoperative values of microscopy sequences providing light intensity profiles or nearly 7 D, before suture adjustment, the average topo- Z-scan graphics, through the z-axis (anteroposterior) dis- graphic cylinder at the end of follow-up was less than 3 D tance calculation between the identified interface layer and after suture removal. Final astigmatism in our series was comparable with or slightly lower than other reported data In our study, IVCM was used to evaluate interface pa- results obtained using and these findings rameters (depth and reflectivity), as well as keratocyte and confirm the efficacy of suture adjustment to control astig- endothelial cell density over a 12-month follow-up. We also matism occurring after as was observed for evaluated the clinical outcome (complication rate and visual Topographic index SRI, a measure of local fluc- acuity) and topographic parameters that could affect visual tuations in central corneal power, and SAI, a measure of the results. Correlations between visual outcome and detected differences in corneal power at every ring over the entire topographic and confocal microscopy interface data also cornea, evaluated at all follow-up examinations, tended to were investigated.
decrease over time. Surface regularity index and SAI values In our series of 46 eyes, 2 DM intraoperative ruptures observed at 12 months corresponded to results reported in occurred during the delamination phase. The larger rupture other studies of and required conversion into PKP. Two other DLKP procedures To our knowledge, this study is the first investigation failed after surgery because of delayed epithelial healing reporting corneal confocal microscopic morphologic and resulting from graft override with severe stromal inflamma- interface features in DLKP. As previously explained for tion and needed subsequent PKP within 5 months of the first LASIK, IVCM allowed easy identification of the interface surgical procedure. The eyes that underwent PKP were layer in DLKP corneas. Deep stromal interface appeared as excluded from data analysis. The incidence of DM split in an atypical stromal layer characterized by discontinuity of our group was 4%, and this frequency is consistent with the tissue and cellular stromal architecture, absence or reduced most favorable outcome reported in previous stud- density of keratocytes, and variable background extracellu- Recent reports from Watson et and from lar reflectivity. Data obtained from averaged Z-scan inter- Shimazaki et aldocumented a DM rupture in 15% of the face depth measurements revealed that the mean depth eyes treated with DLKP with DM exposure. The relatively (distance of the interface from the endothelium) was 60 ␮m lower incidence of DM breaks that occurred in our study (range, 45–74 ␮m) 2 weeks after surgery and remained may be related to the presence of the deepest stromal layers unchanged over the entire follow-up period. These results left undissected above the endothelium, which may protect indicate a reliable depth standardization achievable with the DM from surgical damage during the dissection phase.
technique used. This presumably suggests that significant The logMAR UCVA and BCVA after surgery improved processes of stromal remodeling or deep stromal rethicken- to average values of 0.421 and 0.104, respectively, at 12 ing behind the interface do not take place in DLKP. More- months. The logMAR BCVA was 0.2 or better in 95% of over, in corneas that underwent surgery for keratoconus, the eyes, and 0.1 or better in 70% of eyes at the end of follow-up.
images of residual stromal bed showed a characteristic These findings are similar to those of other studies investigat- presence of hyporeflexive linear striae that were ing visual outcome after DLKP for keratoconus and other present before surgery, which have been shown to be typical anterior corneal pathologic As already men- confocal microscopic features of panstromal involvement in tioned, Sugita and Kondo, in their clinical report on DLKP This aspect further confirms that the deep that included 120 eyes with various corneal diseases other stroma under the interface persists without significant mor- than keratoconus, found that there were no differences in phologic changes, to the extent observable by IVCM. How- BCVA at 12 months between the 2 subgroups of patients ever, it was reported previously that recurrent corneal ecta- Volume 113, Number 8, August 2006 sia after DLKP for keratoconus is a rare but potential could clarify if such scarring phenomena may affect deep late-onset complication that may be related to donor or host stromal interfaces at late postoperative periods. In the and IVCM assessment of preinterface and postint- present study, interface morphologic features at 6 and, par- erface stromal morphologic features and thickness could ticularly, at 12 months after surgery showed a stromal layer improve the understanding of stromal changes in case of with low reflectivity and lack of keratocyte cells. These recurrent keratoconus. Interface morphologic features and features were similar to normal LASIK interface micro- reflectivity showed notable variations during the year of scopic morphologic Anterior and posterior follow-up as opposed to host residual stromal layers. Mean stroma (behind the interface) did not show significant opaci- interface reflectivity values were highest at 2 fication, loss of transparency of the extracellular lamellae, weeks and 1 month and showed a continuous decrease over or relevant keratocyte activation or inflammation. Anterior time, reaching reflectivity levels comparable with normal and posterior keratocyte density were within normal limits rear stromal values at 12 months. In all cases, no (or rare) without significant changes over the 12-month follow-up.
distinguishable keratocytes or myofibroblasts were detected Thus, the only corneal sublayer that evidenced variations in within the interface layers. Interface brightness apparently transparency was the proper donor– host interface. Epithe- was the result of extracellular diffuse and discretely homo- lial morphologic features, nerve fiber regeneration, and but- geneous reflectivity, compatible with edematous fluid accu- ton stromal cellular architecture were not investigated in mulation. This interface extracellular opacity tended to clar- this study, but could be interesting parameters to evaluate in ify over months demonstrating a progressive gain of future studies to understand more thoroughly the micro- transparency of the stroma at the interface layer. The reab- scopic behavior of tissues in DLKP-transplanted corneas.
sorption of interface fluid is the hypothesis that best de- By using the surgical technique presented in our study, a scribes the reduction of interface reflectivity observed in our certain degree of interface opacity occurring within the first study, and apparently no visible scarring features mediated months after surgery was a constant behavior of DLKP by activated keratocytes or myofibroblast were found at the wound healing and should not be considered a complica- interface level during the follow-up period. However, further tion. We did not observe severe or persistent interface in vivo micromorphologic studies, with a longer follow-up, opacity limiting the recovery of visual acuity. Also, slit- BCVARef lectiv ity Figure 5. Graph showing changes over the 12-month follow-up period of 2 main parameters that were correlated with best-corrected visual acuity
(BCVA), reported in logarithm of the minimum angle of resolution units. Interface reflectivity and topographic astigmatism values were reported in a log10
scale. Note that BCVA continued to improve between 3 and 12 months, whereas astigmatism appeared stable In contrast, during the same
period interface reflectivity showed a decrease, statistically significant between 3 and 6 months
Marchini et al 䡠 Deep Lamellar Keratoplasty by Intracorneal Dissection lamp examination allowed the visualization of a mild inter- present significant functional correlation with visual perfor- face haziness mainly up to 3 months after surgery. How- mance after surgery.
ever, biomicroscopic evaluation was not effective in After the 3-month follow-up, no eyes had substantial detecting a fine degree of opacity (at 6 months after surgery, irregular astigmatism (severely interfering with the visual almost all interfaces appeared clear at slit lamp) and did not acuity), but progressive reduction of interface reflectivity permit quantification or grading of this interface parameter was correlated with subsequent BCVA increases up to 1 as objectively as IVCM.
year after surgery. In vivo confocal microscopy is a nonin- Correlation analysis of visual with other measured pa- vasive method for quantitative evaluation of interface trans- rameters showed that interface reflectivity presents a signif- parency in lamellar grafts that could be applied in the other icant negative correlation with BCVA starting from 3 DLKP techniques to find possible differences.
months, when the effect of astigmatism and topographicparameters was reduced. In fact, a significant negative cor-relation was detected between UCVA and topographic astigmatism during the entire follow-up as expected, andbetween UCVA and SRI and SAI up to 1 and 3 months, 1. Shimazaki J. The evolution of lamellar keratoplasty. Curr respectively. As opposed to BCVA, which showed only Opin Ophthalmol 2000;11:217–23.
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This may indicate that interface transparency plays a role in 5. Lyons CJ, McCartney AC, Kirkness CM, et al. Granular visual recovery, which becomes more evident when the ef- corneal dystrophy: visual results and pattern of recurrence fects of other influencing parameters, such as irregular astig- after lamellar or penetrating keratoplasty. Ophthalmology matism, have been controlled. As summarized in average BCVA continued to increase after the stabilization of 6. Benson WH, Goosey CB, Prager TC, Goosey JD. Visual improvement as a function of time after lamellar keratoplasty astigmatism at 3 months, with a parallel decrease of interface for keratoconus. Am J Ophthalmol 1993;116:207–11.
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