LABORATORY SCIENCE
In vitro immunohistochemical and morphological observations of penetrating corneal incisions created by a femtosecond laser used for assisted intraocular lens surgery Wolfgang J. Mayer, MD, FEBO, Oliver K. Klaproth, Dipl-Ing (FH), Fritz H. Hengerer, MD, PhD, Daniel Kook, MD, PhD, FEBO, Martin Dirisamer, MD, FEBO, Siegfried Priglinger, MD, PhD, FEBO, Thomas Kohnen, MD, PhD, FEBO
PURPOSE: To compare inflammatory cell response and morphological aspects of femtosecond laser–created corneal incisions. SETTING: Department of Ophthalmology, Goethe-University, Frankfurt am Main, Germany. DESIGN: Experimental study. METHODS: In 16 of 22 human corneoscleral buttons, clear corneal tunnel incisions were created using a femtosecond laser (Lensx) with 7 mJ laser pulse energy on the outer periphery and manually using a phaco knife on the respective opposite side (180 degrees). In 6 corneas, no treatment was performed (controls). Corneas were then kept in organ culture for 12 or 48 hours, and the inflammatory reaction was evaluated using standard immunofluorescence analyses for monocytes (CD11b) and for dendritic cells (HLA-DR). For morphological analyses and apoptosis, van Gieson staining and terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling was performed. RESULTS: There were no statistically significant differences in inflammatory cell response between femtosecond laser corneal incisions and manually performed incisions. Apoptosis was significantly more pronounced in the femtosecond incisions. The ratio of dendritic cells between femtosecond incisions and manual incisions was 1:2 (12 hours and 48 hours; PZ.07), the ratio of monocytes was 1:2 (12 hours and 48 hours; PZ.08), and the ratio of apoptotic cells was 1:5 (12 hours) and 1:6 (48 hours) (PZ.02). Femtosecond laser incisions showed a more sawtooth-like cutting edge than manual incisions. CONCLUSIONS: Femtosecond laser–created corneal incisions in human corneas showed no differences in inflammatory cell response but a significantly higher cell death rate than manually performed incisions, indicating an upregulated postoperative wound-healing response. Financial Disclosure(s): Proprietary or commercial disclosures are listed after the references. J Cataract Refract Surg 2014; 40:632–638 Q 2014 ASCRS and ESCRS
A new approach in intraocular lens (IOL) surgery is the use of femtosecond lasers to perform anterior capsulotomy and lens fragmentation.1–4 In addition to these main types of IOL surgery, corneal incisions can also be created with a femtosecond laser.1,2,5,6 The advantage of using femtosecond lasers are that they create precisely located and architectured corneal incisions for paracentesis and IOL implantation; they can also be used to perform on-axis placement of the main incision and arcuate relaxing incisions to control surgically induced astigmatism.7–9 632
Q 2014 ASCRS and ESCRS Published by Elsevier Inc.
During any cataract surgery the cornea is injured. Tissue is destroyed and cellular response is involved in corneal wound healing, regardless of whether the incisions are performed manually or with the assistance of a femtosecond laser. The principle of a femtosecond laser, as opposed to manual incision creation, is to create closely spaced microcavitations by photodisruption to separate tissue.10 Some studies11–13 show a cell-mediated immune response after femtosecond laser flap creation in laser in situ keratomileusis (LASIK). The corneal 0886-3350/$ - see front matter http://dx.doi.org/10.1016/j.jcrs.2014.02.015
LABORATORY SCIENCE: IMMUNOCHEMICAL AND MORPHOLOGICAL OBSERVATIONS OF FEMTOSECOND-CREATED PENETRATING INCISIONS
wound-healing response after LASIK performed with the femtosecond laser has been associated with greater postoperative inflammation, mainly in the flap periphery.12,13 The immunologically privileged cornea is endowed with a characteristic population of immune-competent cells, such as antigen-presenting cells and monocytes, that are involved in immune reactions (ie, graft failure after keratoplasty). These cells serve as immune sentinels and are distributed throughout the entire cornea.14–16 In the field of corneal surgery, tissue responses, including cellular effects, are of significant interest in understanding and preventing wound healing–related complications. In addition to the regeneration processes on the cellular level, corneal cell death occurs after injury or surgical treatment and scar formation is introduced. The aim of this study was to compare the cellular effects and morphological aspects of corneal incisions in human corneal donor grafts that are created during cataract surgery. The incisions were performed using a femtosecond laser or manually with a phaco knife. MATERIALS AND METHODS The experimental study was approved by the local ethics committee and adhered to the tenets of the Declaration of Helsinki. It included 22 healthy human sclerocorneal buttons that were kept in organ culture for a maximum of 3 weeks under 37 C until treatment. The organ culture medium contained 25 mL Roswell Park Memorial Institute 1640 culture medium (with 2.0 g/L sodium bicarbonate, without phenol red and without L-glutamine [Biochrome AG]) supplemented with 100 U/mL penicillin G, 100 g/mL streptomycin, 0.25 g/mL amphotericin B (Gibco/Invitrogen Corp.), 2 mM L-glutamine (low endotoxin, Biochrome AG), 25 mM N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (Biochrome AG), and 5% fetal calf serum (Gibco/Invitrogen Corp.).
Submitted: November 11, 2013. Final revision submitted: January 10, 2014. Accepted: February 5, 2014. From the Departments of Ophthalmology, Goethe-University (Mayer, Klaproth, Hengerer, Kohnen), Frankfurt am Main, and Ludwig-Maximilians-University (Mayer, Kook), Munich, Germany; the Department of Ophthalmology (Dirisamer, Priglinger), Allgemeines Krankenhaus Linz, Austria. Ralf Lieberz and Sabine Albrecht, Department of Pathology, Goethe-University, assisted in the sample preparation and staining techniques. Corresponding author: Thomas Kohnen, MD, PhD, FEBO, Department of Ophthalmology, Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. E-mail: [email protected].
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Surgical Technique Sixteen of the 22 corneal buttons were mounted on an artificial anterior chamber (Katena Products, Inc.) for surgical treatment. A primary clear corneal tunnel incision was created on 1 outer periphery using a Lensx femtosecond laser (Alcon Laboratories, Inc.). This 50 kHz femtosecond infrared laser has a pulse width of 600 to 800 fs, a central laser wavelength of 1030 nm, and maximum pulse energy of 15 mJ. Table 1 shows the femtosecond laser parameters used to create the corneal incisions. Corneal applanation was performed using a Softfit interface (Alcon Laboratories, Inc.) consisting of a curved interface (10.8 mm diameter; 8.3 mm curvature) with a vacuum suction ring and a soft contact lens. After successful docking to the mounted corneoscleral button, vacuum suction was confirmed and alignment of the procedure settings for the corneal incisions was performed using spectral-domain optical coherence tomography integrated into the laser system. After manual verification of each procedural step, laser treatment was performed (Figure 1). After laser treatment, a manually performed 2-plane clear corneal tunnel incision was created directly after laser undocking on the opposite side of the same cornea (180 degrees, Figure 2) using a phaco knife with a 2.4 mm cutting width (Clearcut HP2 slit knife, Alcon Laboratories Inc.).
Immunohistochemistry and Terminal Deoxynucleotidyl Transferase Deoxy-UTP-Nick End Labeling Assay After successful creation of the incision, the 16 treated corneas were kept in organ culture for another 12 hours (n Z 8) or 48 hours (n Z 8) to await acute and prolonged cellular response.13,17,18 Although no treatment was performed on the 6 control corneas, they were kept in organ culture for the same timespan (ie, 3 for 12 hours; 3 for 48 hours). Afterward, all corneas were cryofixed at 20 C and were evaluated for an inflammatory or apoptotic cellular response using immunofluorescence staining according to protocols described earlier13,14 for different cell markers as follows: CD11b (Alexa647-anti-CD11b, clone ICRF44 IgG1, 5 mg/mL, eBiosciences Europe) for monocytes and specific HLA-DR (fluorescein isothiocyanate–conjugated anti-HLADR, clone L243, IgG2a, 2.5mg/mL, BD Biosciences Europe) for dendritic cells. The tissue sections were fixed in acetone at room temperature for 5 minutes, air dried for 15 minutes, and then placed in a balanced salt solution before staining. Specific antigens were differentiated through a blocking step using mouse gamma globulin (Jackson Immunoresearch Laboratories, Inc.).
Table 1. Femtosecond laser parameters used for corneal incisions. Parameter Incision width (mm) Pulse energy (mJ) Spot separation (mm) Layer separation (mm) *Per manufacturer's advice
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Value 2.4* 7.0* 4.0 4.0
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LABORATORY SCIENCE: IMMUNOCHEMICAL AND MORPHOLOGICAL OBSERVATIONS OF FEMTOSECOND-CREATED PENETRATING INCISIONS
Figure 1. Morphological aspects of the cutting edge in femtosecond laser corneal specimen (A and B) show a more sawtooth-like pattern than in a manually created incision (C and D), which has a smoother cutting line (original magnification 100, A and C; 400, B and D).
Furthermore, a terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) assay was performed using a standard kit (in situ cell death detection kit, Texas-Red, Roche) to detect apoptosis.13 The TUNEL assay detects the ends of DNA fragments formed during the apoptosis process. To differentiate between nonspecific cells, counterstaining of cryosections was performed using 40 ,6Diamidino-2-phenylindole, dihydrochloride (DAPI) in both treatment groups. Then, all tissue sections were covered with a fluorescent mounting medium (Vectashield, Vector Laboratories). For the morphological evaluation of cutting edges, standard van Gieson staining of samples (Merck Millipore Corp.) was performed.
180-degree manual). Eight sections of each cornea were analyzed and cell counts were averaged. Morphological aspects in van Gieson specimens were obtained using magnifications of 100 and 400.
Statistical Analysis The analysis and cell counts were evaluated using SPSS software (version 21, IBM/SPSS Statistics). The Student t test and Wilcoxon test were used to compare sample means and the number of positive-labeled cells between groups. Variations were expressed as standard errors of the mean. A P value less than 0.05 was considered significant.
RESULTS
Quantitative Analysis A digital confocal microscope (Fluoview 1000, Olympus Corp.) was used to analyze and obtain digital images. Each section was inspected over its entire length using total magnifications of 100, 200, and 400. The expressions of all used assays were recorded. A software-controlled scanning grid (PicED Cora, Jomesa Messsysteme GmbH) was used to count the positive-stained cells in different corneal areas using a method described by Mohan et al.17 For cell quantification, only cells with an illumination threshold of more than 70% in counterstained samples were visualized and counted along the cutting edges. Briefly, the total number of cells in 3 nonoverlapping fullthickness columns extending from the anterior epithelial surface to the posterior stromal surface was manually counted for each specimen. The diameter of each column was the 400 microscopic field. The 3 columns in which counts were performed were aligned along the corneal incisions on each side of the specimen (femtosecond laser versus
Procedures were completed in all groups with no complications. Morphological analysis of van Gieson–stained femtosecond laser incision specimens showed a sawtooth pattern with partial tissue bridges along a regular running cutting edge that did not affect the surrounding tissue (Figure 1, A and B). Bridges were found mainly in the upper anterior corneal limbal area. The manual incisions showed fewer bridges and a smoother cutting line along the edges (Figure 1, C and D). Inflammatory Cell Response There was no statistically significant difference in the number of HLA-DR positive cells, indicating mature dendritic cells, along the cutting edge between the
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in the epithelium and the anterior stroma in both treatment groups (green-stained cells in Figure 2). The ratio of dendritic cells between femtosecond incisions and manually incisions was 1:2 after 12 hours of organ culture (mean 17.3 G 5.1 cells/400 field versus 14.2 G 4.5 cells/400 field) and 1:2 after 48 hours of organ culture (mean 20.3 G 5.6 cells/400 field versus 16.3 G 5.5 cells/400 field) (PZ.07) (Figure 3). In both treatment groups, monocytes (CD11b-positive cells) were found in a lower amount but in a similar cell response as those found in dendritic cells. The CD11b-positive monocytes were mainly seen in the anterior stroma near the cutting edges (red-stained cells in Figure 2). The ratio of CD11b-positive cells between femtosecond incisions and manual incisions was 1:2 after 12 hours of organ culture (mean 12.1 G 4.2 cells/400 field versus 9.7 G 4.5 cells/400 field) and 1:2 after 48 hours of organ culture (mean 15.5 G 4.7 cells/400 field versus 12.9 G 5.2 cells/400 field). However, the differences in cell counts between groups were not statistically significant (PZ.08) (Figure 3).
Figure 2. Distribution of dendritic cells (HLA-DR positive, green cells) and monocytes (CD11b positive, red cells) along the cutting edge of the femtosecond incision (A) and of the manual incision (B) after 12 hours of organ culture after treatment. Counterstaining of nonspecific cells using DAPI technique (blue cells) (original magnification 100).
femtosecond laser incisions and manual incisions after 12 hours and 48 hours of organ culture (PZ.07). All recorded dendritic cells were positive for HLA-DR. Positive-labeled cells were mainly found near the cut edge
Apoptosis All the corneas in which femtosecond laser or manual treatment was performed showed TUNEL-positive cells undergoing apoptosis mainly in the anterior corneal stroma along the cutting edge at 12 hours and 48 hours. In femtosecond incisions, apoptosis was significantly more pronounced along the cutting edge after 12 hours of organ culture than in manual incisions (PZ.02) (red cells in Figure 4, A); apoptotic cell reaction increased during 48 hours of organ culture. In manual incisions, the apoptotic cell reaction was statistically significantly lower but also increased after 48 hours of organ culture (red cells in Figure 4, B). The ratio of apoptotic cells between femtosecond incisions and manual incisions was 1:6 after 12 hours (mean 33.2 G 5.8 cells/400 field
Figure 3. Different inflammatory and cell death quantifications after 12 hours (left) and 48 hours (right) of organ culture after surgical treatment (* Z significant difference in apoptotic cell response between groups, PZ.02; FS Z femtosecond; Tunel Z terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling). J CATARACT REFRACT SURG - VOL 40, APRIL 2014
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LABORATORY SCIENCE: IMMUNOCHEMICAL AND MORPHOLOGICAL OBSERVATIONS OF FEMTOSECOND-CREATED PENETRATING INCISIONS
Figure 4. Distribution of apoptotic cells (TUNEL positive, red cells) along the cutting edge in the femtosecond group (A) and the manual group (B) after treatment and 12 hours of organ culture. Counterstaining of nonspecific cells using DAPI technique (blue cells) (original magnification 100).
versus 21.2 G 6.2 cells/400 field) and 1:5 after 48 hours (mean 40.1 G 6.0 cells/400 field versus 27.3 G 6.7 cells/400 field) (PZ.02) (Figure 3). The controls without treatment showed neither an upregulated specific inflammatory response nor an upregulated cell death reaction after organ culture. DISCUSSION For modern IOL surgery, precise and small corneal incisions are mandatory, not only for foldable IOL
implantation and predictable astigmatism outcomes but also to avoid damaging sensitive structures (eg, corneal endothelium) and for adequate wound closure.19–21 An advantage of the femtosecond laser is the precise setting of parameters; that is, the corneal incision width, form, and length.4,22 This study compared femtosecond laser–created corneal incisions and manually created corneal incisions in morphological aspects and on the cellular level in a human in vitro model. Corneal wound healing after femtosecond laser–assisted surgery includes inflammatory cell infiltration and cell death. In this study, a 50 kHz infrared femtosecond laser (Lensx) was used. We found no significant difference in inflammatory cell response between the femtosecond laser procedures and the manual ones. In contrast, apoptotic cell reaction was more upregulated in the femtosecond laser group. Similar findings have been reported in studies using femtosecond lasers to create flaps in LASIK procedures; however, depending on the laser pulse energy levels, the tissue response and cellular effect might vary.12,13 In our previous study of corneal flap analyses during LASIK,13 there were no significant differences between the femtosecond laser procedure and the manual procedure using a microkeratome. These findings are consistent with other data in the literature.12,17 However, these studies analyzed lasers that create corneal flaps in the anterior stroma. When using a femtosecond laser to create consistent clear corneal incisions, as in cataract or refractive lens surgery, other treatment algorithms and higher laser pulse energy levels are required. For the system used in this study, the manufacturer-recommended pulse energy is 7 mJ. As the present study shows, the upregulated energy level leads to a significantly higher apoptotic cell response. We hypothesize that the higher levels of cell death in contrast to the similar immune-mediated cell infiltration assessed by femtosecond laser are attributable to a direct thermal-induced energy-related apoptotic effect on cell death associated with the femtosecond laser. In the clinical environment, this inflammatory reaction can be suppressed with topical corticosteroids during the early postoperative phase. Cell apoptosis and the associated release of intracellular components into the tissue have a direct chemotactically active effect on inflammatory cells and may promote inflammatory cell infiltration. In addition, the extent of damage to the corneal epithelium and stroma produced is a further trigger of inflammation. Changes in energy settings in femtosecond laser models result in corneal damage, release of epithelialderived cytokines, production of keratocyte-derived chemokines, and inflammatory cell infiltration, especially near the cutting edge.11,12,23
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LABORATORY SCIENCE: IMMUNOCHEMICAL AND MORPHOLOGICAL OBSERVATIONS OF FEMTOSECOND-CREATED PENETRATING INCISIONS
A limitation of our study is the in vitro human model used. We chose this model because significantly more animal data on corneal cell response after laser treatment are available in the literature. Although culture condition may modulate the effect on cells after any sort of treatment, we are aware that a nonspecific apoptotic and inflammatory cell response might occur. Therefore, the untreated control group was analyzed. Further optimization of femtosecond laser technology is required to achieve optimum cutting procedures with minimal tissue burden in the cornea. In conclusion, our in vitro series of human corneas confirms existing animal data on corneal cell behavior and further implicates comparable inflammatory but stronger apoptotic cell reaction in femtosecond laser– created human corneal incisions than in manually created incisions.
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WHAT WAS KNOWN Femtosecond lasers produce accurate corneal flaps. The design of the patient interface is important to the outcomes of the procedure. WHAT THIS PAPER ADDS Human corneal incisions produced by a femtosecond laser during assisted IOL surgery showed a sawtooth pattern along a regular running cutting edge that did not affect the surrounding tissue. Femtosecond laser–created corneal incisions showed no differences in inflammatory cell response but a significantly higher cell death rate than manually performed incisions, indicating an upregulated postoperative wound-healing response.
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FINANCIAL DISCLOSURE(S) Dr. Mayer received travel reimbursements and/or lecture fees from Allergan and is a consultant to Allergan and Carl Zeiss Meditec. Mr. Klaproth received travel reimbursements and/or lecture fees from Alcon €te GmbH, Laboratories, Inc., Carl Zeiss Meditec, Oculus Optikgera and Rayner Intraocular Lenses Ltd, and is a consultant to RTI Health Solutions. Dr. Kohnen received travel reimbursements and/or lecture fees from Alcon Laboratories, Inc., Abbott Medical Optics, Inc., Bausch & Lomb, Carl Zeiss Meditec AG, Neoptics AG, Rayner Intraocular Lenses Ltd., and Schwind eye-tech-solutions GmbH and Co. KG., and is a consultant to Alcon Laboratories, Inc., Carl Zeiss Meditec AG, Thieme Compliance GmbH, Rayner Intraocular Lenses Ltd, and Schwind eye-tech-solutions GmbH and Co. KG. No other author has a financial or proprietary interest in any material or method mentioned.
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REFERENCES 1. Roberts TV, Lawless M, Chan CCK, Jacobs M, Ng D, Bali SJ, Hodge C, Sutton G. Femtosecond laser cataract surgery:
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technology and clinical practice. Clin Exp Ophthalmol 2013; 41:180–186 Roberts TV, Lawless M, Bali SJ, Hodge C, Sutton G. Surgical outcomes and safety of femtosecond laser cataract surgery: a prospective study of 1500 consecutive cases. Ophthalmology 2013; 120:227–233 Abell RG, Kerr NM, Vote BJ. Toward zero effective phacoemulsification time using femtosecond laser pretreatment. Ophthalmology 2013; 120:942–948 Conrad-Hengerer I, Hengerer FH, Schultz T, Dick HB. Effect of femtosecond laser fragmentation on effective phacoemulsification time in cataract surgery. J Refract Surg 2012; 28:879–883 Abell RG, Kerr NM, Vote BJ. Femtosecond laser-assisted cataract surgery compared with conventional cataract surgery. Clin Exp Ophthalmol 2013; 41:455–462 Nagy Z, Takacs A, Filkorn T, Sarayba M. Initial clinical evaluation of an intraocular femtosecond laser in cataract surgery. J Refract Surg 2009; 25:1053–1060 Wetterstrand O, Holopainen JM, Krootila K. Treatment of postoperative keratoplasty astigmatism using femtosecond laser– assisted intrastromal relaxing incisions. J Refract Surg 2013; 29:378–382 Kohnen T. Astigmatism measurements for cataract and refractive surgery [editorial]. J Cataract Refract Surg 2012; 38:2065 €hren J, Klaproth OK, Bauch AS, Derhartunian V, Kook D, Bu Kohnen T. Astigmatische Keratotomie mit dem Femtosekundenlaser. Korrektur hoher Astigmatismen nach Keratoplastik [Astigmatic keratotomy with the femtosecond laser. Correction of high astigmatisms after keratoplasty]. Ophthalmologe 2011; 108:143–150 €ttman G, Paltauf G. Mechanisms of femtoVogel A, Noack J, Hu second laser nanosurgery of cells and tissues. Appl Phys B Lasers Opt 2005; 81:1015–1047. Available at: https://www5.uva. es/guia_docente/uploads/2011/363/50537/1/Documento1.pdf. Accessed February 5, 2014 €hren J, Bug R, Ohrloff C, Deller T. Meltendorf C, Burbach GJ, Bu Corneal femtosecond laser keratotomy results in isolated stromal injury and favorable wound-healing response. Invest Ophthalmol Vis Sci 2007; 48:2068–2075. Available at: http://www. iovs.org/cgi/reprint/48/5/2068. Accessed February 5, 2014 Netto MV, Mohan RR, Medeiros FW, Dupps WJ Jr, Sinha S, Krueger RR, Stapleton WM, Rayborn M, Suto C, Wilson SE. Femtosecond laser and microkeratome corneal flaps: Comparison of stromal wound healing and inflammation. J Refract Surg 2007; 23:667–676. Available at: http://www.ncbi.nlm.nih.gov/ pmc/articles/PMC2698458/pdf/nihms118773.pdf. Accessed February 5, 2014 Mayer WJ, Grueterich M, Wolf AH, Lackerbauer CA, Eibl K, Kampik A, Kook D. Corneal cell response after flap creation using a mechanical microkeratome or a 200 kHz femtosecond laser. J Cataract Refract Surg 2013; 39:1088–1092 Mayer WJ, Irschick UM, Moser P, Wurm M, Huemer HP, Romani N, Irschick EU. Characterization of antigen-presenting cells in fresh and cultured human corneas using novel dendritic cell markers. Invest Ophthalmol Vis Sci 2007; 48:4459–4467. Available at: http://www.iovs.org/content/48/10/4459.full.pdf. Accessed February 5, 2014 Yamagami S, Ebihara N, Usui T, Yokoo S, Amano S. Bone marrow-derived cells in normal human corneal stroma. Arch Ophthalmol 2006; 124:62–69. Available at: http://archopht.jamanetwork.com/data/Journals/OPHTH/9950/ELS50013.pdf. Accessed February 5, 2014 Hamrah P, Liu Y, Zhang Q, Dana MR. Alterations in corneal stromal dendritic cell phenotype and distribution in
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inflammation. Arch Ophthalmol 2003; 121:1132–1140. Correction 1555. Available at: http://archopht.jamanetwork.com/data/ Journals/OPHTH/9911/ELS20040.pdf. Correction available at: http://archopht.jamanetwork.com/data/Journals/OPHTH/9915/ ECX30006.pdf. Accessed both February 5, 2014 Mohan RR, Hutcheon AE, Choi R, Hong JW, Lee JS, Mohan RR, sio R Jr, Zieske JD, Wilson SE. Apoptosis, necrosis, proAmbro liferation, and myofibroblast generation in the stroma following LASIK and PRK. Exp Eye Res 2003; 76:71–87 Wilson SE, Mohan RR, Hong J, Lee J, Choi R, Liu JJ, Mohan RR. Apoptosis in the cornea in response to epithelial injury: significance to wound healing and dry eye. Adv Exp Med Biol 2002; 506:821–826 Kohnen T, Lambert RJ, Koch DD. Incision sizes for foldable intraocular lenses. Ophthalmology 1997; 104:1277–1286 €ller M, Kohnen T. Inzisionen fu €r die biaxiale und koaxiale Mu mikroinzisionale Kataraktchirurgie [Incisions for biaxial and
coaxial microincision cataract surgery]. Ophthalmologe 2010; 107:108–115 21. Vasavada AR, Johar K Sr, Praveen MR, Vasavada VA, Arora AI. Histomorphological and immunofluorescence evaluation of clear corneal incisions after microcoaxial phacoemulsification with 2.2 mm and 1.8 mm systems. J Cataract Refract Surg 2013; 39:617–623 22. Reddy KP, Kandulla J, Auffarth GU. Effectiveness and safety of femtosecond laser–assisted lens fragmentation and anterior capsulotomy versus the manual technique in cataract surgery. J Cataract Refract Surg 2013; 39:1297–1306 23. Hong J-W, Liu JJ, Lee J-S, Mohan RR, Mohan RR, Woods DJ, He Y-G, Wilson SE. Proinflammatory chemokine induction in keratocytes and inflammatory cell infiltration into the cornea. Invest Ophthalmol Vis Sci 2001; 42:2795–2803. Available at: http://www.iovs.org/cgi/reprint/42/12/2795.pdf. Accessed February 5, 2014
J CATARACT REFRACT SURG - VOL 40, APRIL 2014
In vitro immunohistochemical and morphological observations of penetrating corneal incisions created by a femtosecond laser used for assisted intraocular lens surgery Wolfgang J. Mayer, MD, FEBO, Oliver K. Klaproth, Dipl-Ing (FH), Fritz H. Hengerer, MD, PhD, Daniel Kook, MD, PhD, FEBO, Martin Dirisamer, MD, FEBO, Siegfried Priglinger, MD, PhD, FEBO, Thomas Kohnen, MD, PhD, FEBO
PURPOSE: To compare inflammatory cell response and morphological aspects of femtosecond laser–created corneal incisions. SETTING: Department of Ophthalmology, Goethe-University, Frankfurt am Main, Germany. DESIGN: Experimental study. METHODS: In 16 of 22 human corneoscleral buttons, clear corneal tunnel incisions were created using a femtosecond laser (Lensx) with 7 mJ laser pulse energy on the outer periphery and manually using a phaco knife on the respective opposite side (180 degrees). In 6 corneas, no treatment was performed (controls). Corneas were then kept in organ culture for 12 or 48 hours, and the inflammatory reaction was evaluated using standard immunofluorescence analyses for monocytes (CD11b) and for dendritic cells (HLA-DR). For morphological analyses and apoptosis, van Gieson staining and terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling was performed. RESULTS: There were no statistically significant differences in inflammatory cell response between femtosecond laser corneal incisions and manually performed incisions. Apoptosis was significantly more pronounced in the femtosecond incisions. The ratio of dendritic cells between femtosecond incisions and manual incisions was 1:2 (12 hours and 48 hours; PZ.07), the ratio of monocytes was 1:2 (12 hours and 48 hours; PZ.08), and the ratio of apoptotic cells was 1:5 (12 hours) and 1:6 (48 hours) (PZ.02). Femtosecond laser incisions showed a more sawtooth-like cutting edge than manual incisions. CONCLUSIONS: Femtosecond laser–created corneal incisions in human corneas showed no differences in inflammatory cell response but a significantly higher cell death rate than manually performed incisions, indicating an upregulated postoperative wound-healing response. Financial Disclosure(s): Proprietary or commercial disclosures are listed after the references. J Cataract Refract Surg 2014; 40:632–638 Q 2014 ASCRS and ESCRS
A new approach in intraocular lens (IOL) surgery is the use of femtosecond lasers to perform anterior capsulotomy and lens fragmentation.1–4 In addition to these main types of IOL surgery, corneal incisions can also be created with a femtosecond laser.1,2,5,6 The advantage of using femtosecond lasers are that they create precisely located and architectured corneal incisions for paracentesis and IOL implantation; they can also be used to perform on-axis placement of the main incision and arcuate relaxing incisions to control surgically induced astigmatism.7–9 632
Q 2014 ASCRS and ESCRS Published by Elsevier Inc.
During any cataract surgery the cornea is injured. Tissue is destroyed and cellular response is involved in corneal wound healing, regardless of whether the incisions are performed manually or with the assistance of a femtosecond laser. The principle of a femtosecond laser, as opposed to manual incision creation, is to create closely spaced microcavitations by photodisruption to separate tissue.10 Some studies11–13 show a cell-mediated immune response after femtosecond laser flap creation in laser in situ keratomileusis (LASIK). The corneal 0886-3350/$ - see front matter http://dx.doi.org/10.1016/j.jcrs.2014.02.015
LABORATORY SCIENCE: IMMUNOCHEMICAL AND MORPHOLOGICAL OBSERVATIONS OF FEMTOSECOND-CREATED PENETRATING INCISIONS
wound-healing response after LASIK performed with the femtosecond laser has been associated with greater postoperative inflammation, mainly in the flap periphery.12,13 The immunologically privileged cornea is endowed with a characteristic population of immune-competent cells, such as antigen-presenting cells and monocytes, that are involved in immune reactions (ie, graft failure after keratoplasty). These cells serve as immune sentinels and are distributed throughout the entire cornea.14–16 In the field of corneal surgery, tissue responses, including cellular effects, are of significant interest in understanding and preventing wound healing–related complications. In addition to the regeneration processes on the cellular level, corneal cell death occurs after injury or surgical treatment and scar formation is introduced. The aim of this study was to compare the cellular effects and morphological aspects of corneal incisions in human corneal donor grafts that are created during cataract surgery. The incisions were performed using a femtosecond laser or manually with a phaco knife. MATERIALS AND METHODS The experimental study was approved by the local ethics committee and adhered to the tenets of the Declaration of Helsinki. It included 22 healthy human sclerocorneal buttons that were kept in organ culture for a maximum of 3 weeks under 37 C until treatment. The organ culture medium contained 25 mL Roswell Park Memorial Institute 1640 culture medium (with 2.0 g/L sodium bicarbonate, without phenol red and without L-glutamine [Biochrome AG]) supplemented with 100 U/mL penicillin G, 100 g/mL streptomycin, 0.25 g/mL amphotericin B (Gibco/Invitrogen Corp.), 2 mM L-glutamine (low endotoxin, Biochrome AG), 25 mM N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (Biochrome AG), and 5% fetal calf serum (Gibco/Invitrogen Corp.).
Submitted: November 11, 2013. Final revision submitted: January 10, 2014. Accepted: February 5, 2014. From the Departments of Ophthalmology, Goethe-University (Mayer, Klaproth, Hengerer, Kohnen), Frankfurt am Main, and Ludwig-Maximilians-University (Mayer, Kook), Munich, Germany; the Department of Ophthalmology (Dirisamer, Priglinger), Allgemeines Krankenhaus Linz, Austria. Ralf Lieberz and Sabine Albrecht, Department of Pathology, Goethe-University, assisted in the sample preparation and staining techniques. Corresponding author: Thomas Kohnen, MD, PhD, FEBO, Department of Ophthalmology, Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. E-mail: [email protected].
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Surgical Technique Sixteen of the 22 corneal buttons were mounted on an artificial anterior chamber (Katena Products, Inc.) for surgical treatment. A primary clear corneal tunnel incision was created on 1 outer periphery using a Lensx femtosecond laser (Alcon Laboratories, Inc.). This 50 kHz femtosecond infrared laser has a pulse width of 600 to 800 fs, a central laser wavelength of 1030 nm, and maximum pulse energy of 15 mJ. Table 1 shows the femtosecond laser parameters used to create the corneal incisions. Corneal applanation was performed using a Softfit interface (Alcon Laboratories, Inc.) consisting of a curved interface (10.8 mm diameter; 8.3 mm curvature) with a vacuum suction ring and a soft contact lens. After successful docking to the mounted corneoscleral button, vacuum suction was confirmed and alignment of the procedure settings for the corneal incisions was performed using spectral-domain optical coherence tomography integrated into the laser system. After manual verification of each procedural step, laser treatment was performed (Figure 1). After laser treatment, a manually performed 2-plane clear corneal tunnel incision was created directly after laser undocking on the opposite side of the same cornea (180 degrees, Figure 2) using a phaco knife with a 2.4 mm cutting width (Clearcut HP2 slit knife, Alcon Laboratories Inc.).
Immunohistochemistry and Terminal Deoxynucleotidyl Transferase Deoxy-UTP-Nick End Labeling Assay After successful creation of the incision, the 16 treated corneas were kept in organ culture for another 12 hours (n Z 8) or 48 hours (n Z 8) to await acute and prolonged cellular response.13,17,18 Although no treatment was performed on the 6 control corneas, they were kept in organ culture for the same timespan (ie, 3 for 12 hours; 3 for 48 hours). Afterward, all corneas were cryofixed at 20 C and were evaluated for an inflammatory or apoptotic cellular response using immunofluorescence staining according to protocols described earlier13,14 for different cell markers as follows: CD11b (Alexa647-anti-CD11b, clone ICRF44 IgG1, 5 mg/mL, eBiosciences Europe) for monocytes and specific HLA-DR (fluorescein isothiocyanate–conjugated anti-HLADR, clone L243, IgG2a, 2.5mg/mL, BD Biosciences Europe) for dendritic cells. The tissue sections were fixed in acetone at room temperature for 5 minutes, air dried for 15 minutes, and then placed in a balanced salt solution before staining. Specific antigens were differentiated through a blocking step using mouse gamma globulin (Jackson Immunoresearch Laboratories, Inc.).
Table 1. Femtosecond laser parameters used for corneal incisions. Parameter Incision width (mm) Pulse energy (mJ) Spot separation (mm) Layer separation (mm) *Per manufacturer's advice
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Value 2.4* 7.0* 4.0 4.0
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Figure 1. Morphological aspects of the cutting edge in femtosecond laser corneal specimen (A and B) show a more sawtooth-like pattern than in a manually created incision (C and D), which has a smoother cutting line (original magnification 100, A and C; 400, B and D).
Furthermore, a terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) assay was performed using a standard kit (in situ cell death detection kit, Texas-Red, Roche) to detect apoptosis.13 The TUNEL assay detects the ends of DNA fragments formed during the apoptosis process. To differentiate between nonspecific cells, counterstaining of cryosections was performed using 40 ,6Diamidino-2-phenylindole, dihydrochloride (DAPI) in both treatment groups. Then, all tissue sections were covered with a fluorescent mounting medium (Vectashield, Vector Laboratories). For the morphological evaluation of cutting edges, standard van Gieson staining of samples (Merck Millipore Corp.) was performed.
180-degree manual). Eight sections of each cornea were analyzed and cell counts were averaged. Morphological aspects in van Gieson specimens were obtained using magnifications of 100 and 400.
Statistical Analysis The analysis and cell counts were evaluated using SPSS software (version 21, IBM/SPSS Statistics). The Student t test and Wilcoxon test were used to compare sample means and the number of positive-labeled cells between groups. Variations were expressed as standard errors of the mean. A P value less than 0.05 was considered significant.
RESULTS
Quantitative Analysis A digital confocal microscope (Fluoview 1000, Olympus Corp.) was used to analyze and obtain digital images. Each section was inspected over its entire length using total magnifications of 100, 200, and 400. The expressions of all used assays were recorded. A software-controlled scanning grid (PicED Cora, Jomesa Messsysteme GmbH) was used to count the positive-stained cells in different corneal areas using a method described by Mohan et al.17 For cell quantification, only cells with an illumination threshold of more than 70% in counterstained samples were visualized and counted along the cutting edges. Briefly, the total number of cells in 3 nonoverlapping fullthickness columns extending from the anterior epithelial surface to the posterior stromal surface was manually counted for each specimen. The diameter of each column was the 400 microscopic field. The 3 columns in which counts were performed were aligned along the corneal incisions on each side of the specimen (femtosecond laser versus
Procedures were completed in all groups with no complications. Morphological analysis of van Gieson–stained femtosecond laser incision specimens showed a sawtooth pattern with partial tissue bridges along a regular running cutting edge that did not affect the surrounding tissue (Figure 1, A and B). Bridges were found mainly in the upper anterior corneal limbal area. The manual incisions showed fewer bridges and a smoother cutting line along the edges (Figure 1, C and D). Inflammatory Cell Response There was no statistically significant difference in the number of HLA-DR positive cells, indicating mature dendritic cells, along the cutting edge between the
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in the epithelium and the anterior stroma in both treatment groups (green-stained cells in Figure 2). The ratio of dendritic cells between femtosecond incisions and manually incisions was 1:2 after 12 hours of organ culture (mean 17.3 G 5.1 cells/400 field versus 14.2 G 4.5 cells/400 field) and 1:2 after 48 hours of organ culture (mean 20.3 G 5.6 cells/400 field versus 16.3 G 5.5 cells/400 field) (PZ.07) (Figure 3). In both treatment groups, monocytes (CD11b-positive cells) were found in a lower amount but in a similar cell response as those found in dendritic cells. The CD11b-positive monocytes were mainly seen in the anterior stroma near the cutting edges (red-stained cells in Figure 2). The ratio of CD11b-positive cells between femtosecond incisions and manual incisions was 1:2 after 12 hours of organ culture (mean 12.1 G 4.2 cells/400 field versus 9.7 G 4.5 cells/400 field) and 1:2 after 48 hours of organ culture (mean 15.5 G 4.7 cells/400 field versus 12.9 G 5.2 cells/400 field). However, the differences in cell counts between groups were not statistically significant (PZ.08) (Figure 3).
Figure 2. Distribution of dendritic cells (HLA-DR positive, green cells) and monocytes (CD11b positive, red cells) along the cutting edge of the femtosecond incision (A) and of the manual incision (B) after 12 hours of organ culture after treatment. Counterstaining of nonspecific cells using DAPI technique (blue cells) (original magnification 100).
femtosecond laser incisions and manual incisions after 12 hours and 48 hours of organ culture (PZ.07). All recorded dendritic cells were positive for HLA-DR. Positive-labeled cells were mainly found near the cut edge
Apoptosis All the corneas in which femtosecond laser or manual treatment was performed showed TUNEL-positive cells undergoing apoptosis mainly in the anterior corneal stroma along the cutting edge at 12 hours and 48 hours. In femtosecond incisions, apoptosis was significantly more pronounced along the cutting edge after 12 hours of organ culture than in manual incisions (PZ.02) (red cells in Figure 4, A); apoptotic cell reaction increased during 48 hours of organ culture. In manual incisions, the apoptotic cell reaction was statistically significantly lower but also increased after 48 hours of organ culture (red cells in Figure 4, B). The ratio of apoptotic cells between femtosecond incisions and manual incisions was 1:6 after 12 hours (mean 33.2 G 5.8 cells/400 field
Figure 3. Different inflammatory and cell death quantifications after 12 hours (left) and 48 hours (right) of organ culture after surgical treatment (* Z significant difference in apoptotic cell response between groups, PZ.02; FS Z femtosecond; Tunel Z terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling). J CATARACT REFRACT SURG - VOL 40, APRIL 2014
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Figure 4. Distribution of apoptotic cells (TUNEL positive, red cells) along the cutting edge in the femtosecond group (A) and the manual group (B) after treatment and 12 hours of organ culture. Counterstaining of nonspecific cells using DAPI technique (blue cells) (original magnification 100).
versus 21.2 G 6.2 cells/400 field) and 1:5 after 48 hours (mean 40.1 G 6.0 cells/400 field versus 27.3 G 6.7 cells/400 field) (PZ.02) (Figure 3). The controls without treatment showed neither an upregulated specific inflammatory response nor an upregulated cell death reaction after organ culture. DISCUSSION For modern IOL surgery, precise and small corneal incisions are mandatory, not only for foldable IOL
implantation and predictable astigmatism outcomes but also to avoid damaging sensitive structures (eg, corneal endothelium) and for adequate wound closure.19–21 An advantage of the femtosecond laser is the precise setting of parameters; that is, the corneal incision width, form, and length.4,22 This study compared femtosecond laser–created corneal incisions and manually created corneal incisions in morphological aspects and on the cellular level in a human in vitro model. Corneal wound healing after femtosecond laser–assisted surgery includes inflammatory cell infiltration and cell death. In this study, a 50 kHz infrared femtosecond laser (Lensx) was used. We found no significant difference in inflammatory cell response between the femtosecond laser procedures and the manual ones. In contrast, apoptotic cell reaction was more upregulated in the femtosecond laser group. Similar findings have been reported in studies using femtosecond lasers to create flaps in LASIK procedures; however, depending on the laser pulse energy levels, the tissue response and cellular effect might vary.12,13 In our previous study of corneal flap analyses during LASIK,13 there were no significant differences between the femtosecond laser procedure and the manual procedure using a microkeratome. These findings are consistent with other data in the literature.12,17 However, these studies analyzed lasers that create corneal flaps in the anterior stroma. When using a femtosecond laser to create consistent clear corneal incisions, as in cataract or refractive lens surgery, other treatment algorithms and higher laser pulse energy levels are required. For the system used in this study, the manufacturer-recommended pulse energy is 7 mJ. As the present study shows, the upregulated energy level leads to a significantly higher apoptotic cell response. We hypothesize that the higher levels of cell death in contrast to the similar immune-mediated cell infiltration assessed by femtosecond laser are attributable to a direct thermal-induced energy-related apoptotic effect on cell death associated with the femtosecond laser. In the clinical environment, this inflammatory reaction can be suppressed with topical corticosteroids during the early postoperative phase. Cell apoptosis and the associated release of intracellular components into the tissue have a direct chemotactically active effect on inflammatory cells and may promote inflammatory cell infiltration. In addition, the extent of damage to the corneal epithelium and stroma produced is a further trigger of inflammation. Changes in energy settings in femtosecond laser models result in corneal damage, release of epithelialderived cytokines, production of keratocyte-derived chemokines, and inflammatory cell infiltration, especially near the cutting edge.11,12,23
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A limitation of our study is the in vitro human model used. We chose this model because significantly more animal data on corneal cell response after laser treatment are available in the literature. Although culture condition may modulate the effect on cells after any sort of treatment, we are aware that a nonspecific apoptotic and inflammatory cell response might occur. Therefore, the untreated control group was analyzed. Further optimization of femtosecond laser technology is required to achieve optimum cutting procedures with minimal tissue burden in the cornea. In conclusion, our in vitro series of human corneas confirms existing animal data on corneal cell behavior and further implicates comparable inflammatory but stronger apoptotic cell reaction in femtosecond laser– created human corneal incisions than in manually created incisions.
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WHAT WAS KNOWN Femtosecond lasers produce accurate corneal flaps. The design of the patient interface is important to the outcomes of the procedure. WHAT THIS PAPER ADDS Human corneal incisions produced by a femtosecond laser during assisted IOL surgery showed a sawtooth pattern along a regular running cutting edge that did not affect the surrounding tissue. Femtosecond laser–created corneal incisions showed no differences in inflammatory cell response but a significantly higher cell death rate than manually performed incisions, indicating an upregulated postoperative wound-healing response.
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FINANCIAL DISCLOSURE(S) Dr. Mayer received travel reimbursements and/or lecture fees from Allergan and is a consultant to Allergan and Carl Zeiss Meditec. Mr. Klaproth received travel reimbursements and/or lecture fees from Alcon €te GmbH, Laboratories, Inc., Carl Zeiss Meditec, Oculus Optikgera and Rayner Intraocular Lenses Ltd, and is a consultant to RTI Health Solutions. Dr. Kohnen received travel reimbursements and/or lecture fees from Alcon Laboratories, Inc., Abbott Medical Optics, Inc., Bausch & Lomb, Carl Zeiss Meditec AG, Neoptics AG, Rayner Intraocular Lenses Ltd., and Schwind eye-tech-solutions GmbH and Co. KG., and is a consultant to Alcon Laboratories, Inc., Carl Zeiss Meditec AG, Thieme Compliance GmbH, Rayner Intraocular Lenses Ltd, and Schwind eye-tech-solutions GmbH and Co. KG. No other author has a financial or proprietary interest in any material or method mentioned.
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