ClinicalStudy - Hindawi Publishing...

Clinical StudyAnterior Capsule of the Lens Comparison ofMorphological Properties and Apoptosis Induction followingFLACS and Standard Phacoemulsification Surgery

Alessandra Pisciotta 12 Michele DeMaria 13 Tommaso Verdina1

Elisa Fornasari1 Anto de Pol 2 and GianMaria Cavallini 1

1 Institute of Ophthalmology University of Modena and Reggio Emilia Modena Italy2Department of Surgery Medicine Dentistry and Morphological Sciences University of Modena and Reggio Emilia Modena Italy3Clinical and Experimental Medicine PhD Program University of Modena and Reggio Emilia Modena Italy

Correspondence should be addressed to Alessandra Pisciotta alessandrapisciottaunimoreit andMichele De Maria micheledemaria86gmailcom

Received 12 October 2017 Accepted 17 December 2017 Published 11 January 2018

Academic Editor Gaurav Prakash

Copyright copy 2018 Alessandra Pisciotta et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Purpose Comparative evaluation of morphological features of anterior capsules and apoptosis induction in epithelial cells afterfemtosecond laser-assisted cataract surgery (FLACS) and standard phacoemulsification surgery Methods Group 1 30 FLACSanterior capsulotomies and Group 2 30 manual anterior continuous curvilinear capsulorhexes All patients were operated on bythe same experienced surgeon Morphological features of the anterior capsules and apoptosis induction in epithelial cells wereevaluatedResults All patients revealed a significantmean best-corrected visual acuity (BCVA) improvement 3months after surgeryand no major intraoperative nor postoperative complications occurred The capsular epithelium appeared to be preserved in bothgroups Scanning electronmicroscopy analysis revealed irregular saw-tooth shaped edges in capsules fromGroup 1whereas capsulesfrom Group 2 showed regular and smooth edges A statistically significant higher expression of the downstream apoptotic effectorcleaved caspase 3 was observed in Group 1 Conclusions The saw-tooth appearance was likely due to the progressive sequenceof laser pulses on the capsule The low energyhigh frequency properties of the laser pulse combined with an overlapped pulsepattern resulted in highly continuous morphology of capsule edgesThe higher apoptosis induction in FLACS group might be dueto photodisruption-dependent plasma generation and formation of cavitation bubbles

1 Introduction

Cataract surgery is the most performed surgical interventionworldwide with an increasing number of surgical proceduresup to 24 million per year according to WHO estimations [1]

For several years the phacoemulsification technique hasbeen the standard method for cataract extraction and thecontinuous technical improvements such as the clear cornealmicroincisions the use of foldable intraocular lenses andthe application of lower ultrasound energy for the frag-mentation of the lens have made cataract surgery efficientand safe The bimanual microincision cataract surgery (B-MICS) is a minimally invasive variant of the traditionalcoaxial phacoemulsification which allowing clear corneal

microincisions of 14mm uses a sleeveless phaco tip with aseparated irrigating chopper that grants a higher stability ofthe anterior chamber and the possibility of using both rightand left hands during the surgical procedure [2 3]

The latest update in cataract surgery was the introductionof femtosecond laser technology Starting from femtosecondlasers firstly introduced for corneal surgery with the aimof producing corneal flaps for refractive surgery most ofthe newest generation femtosecond lasers operate in cataractsurgery at a repetition rate up to 160 kHz combined with anenergy range of maximum 10ndash15 120583J [4]

Based on the photodisruption principles such types oflasers allow us to optimally standardize some of the most dif-ficult parts of the procedure including capsulotomy nuclear

HindawiBioMed Research InternationalVolume 2018 Article ID 7242837 8 pageshttpsdoiorg10115520187242837

2 BioMed Research International

fragmentation and corneal incisions besides providingmoreaccurate and reproducible results when compared to manualapproaches [4]

Until the advent of femtosecond laser technology manualcontinuous curvilinear capsulorhexis (mCCC) has been thestandard method for the opening of anterior capsule incataract surgery or refractive lens replacement Femtosecondlaser assisted cataract surgery (FLACS) has now revolution-ized this important surgical step creating predictable andcurvilinear capsulotomies which result in improved capsule-intraocular lens (IOL) overlap and better IOL centration andposition than with the manual technique In particular pre-vious findings demonstrated that FLACS capsulotomies havea repeatable and precise size are centered and consequentlyprovide better IOL centration and reduced probability of IOLtilt [5ndash9]

Several studies have examined the morphology of theedges and the smoothness of FLACS capsulotomies whichare created by specific laser wavelength pulse energy pulseduration pulse pattern repetition rate and spot size [4]Preliminary analyses of the capsules showed a postage-stamp aspect with a significant roughness likely caused byseveral factors including the laser energy used to create thecapsulotomies [10]

Moreover posterior capsule opacification (PCO) is oneof the most recurring long-term complications of cataractsurgery Many studies have been conducted so far in orderto define which factors may influence the developmentof PCO including the IOL design the administration oftherapeutic agents the surgical technique itself and othersurgical procedures to clean the posterior capsule [11 12]

As reported in the literature lens capsule is exposed tomechanical or photodisruptive damage when either capsu-lorhexis or capsulotomy is performed with consequent deathof the epithelial cellsThe hypothesis is that femtosecond lasermight reasonably induce apoptosis to a higher extent

The aim of this study was to evaluate morphologicalfeatures of the human anterior lens capsule even at theultrastructural level following cataract surgery performedthrough manual technique and FLACS respectively Fur-thermore we investigated whether apoptosis was inducedin monolayered epithelium beneath the anterior capsulefollowing the two surgical approaches

2 Materials and Methods

The experimental study was approved by the Human Re-search Ethics Committee of the University of Modena andReggio Emilia (Modena Italy) and was conducted in com-pliance with the Declaration of Helsinki at the Institute ofOphthalmology University of Modena and Reggio EmiliaPatients with cataract LOCS III grade 2 or 3 and a correctdistance visual acuity of 510 or worse were included inthe study Patients were excluded in case of ocular traumapseudoexfoliation syndrome or other ocular comorbiditiesThe study was performed after written consent of the patientsundergoing either traditional manual cataract extraction (B-MICS) or femtosecond laser assisted cataract surgery byusing the femtosecond laser Ziemer LDV Z8 All the surgical

procedures were performed by the same experienced surgeon(GMC)

21 Surgical Techniques A consistent mydriasis was obtainedwith instillation of 10 phenylephrine and 1 cyclopentolateand a locoregional anaesthesia with peribulbar block wasperformed (15mL of lidocaine 2 and 15mL of bupivacaine05)

211 FSL-Assisted Technique We carefully applied a dispos-able suction ring to the eye centered over the limbus Thesuction ring was filled with balanced salt solution (BSS)in order to create a liquid interface and the mobile armof the laser system was docked over the corneal apexThe ocular structures were shown by the integrated opti-cal coherence tomography (OCT) system and treatmentparameters were determined in a customized manner byusing the laser platform settings wizard The energy andfrequency laser pulse settings were 900mW 1MHz forcapsulotomy 950ndash1000mW 2MHz for phacofragmentation1200ndash1300mW 2MHz for corneal incisions Laser treatmentstarted with lens fragmentation with an eight-piece pie-cut pattern followed by an anterior capsulotomy of 52mmdiameter Finally the laser performed two 14mm cornealmicroincisions at 10 orsquoclock and 2 orsquoclock respectively Atthe end the suction ring was removed from the eye surfaceto proceed with the phaco procedure The surgeon checkedthe patency of the CCIs using a smooth spatula first with aperpendicular inclination to the limbus inclination and thenwith gentle movements along the incision in order to open itcompletely from side to side The anterior chamber was filledwith ocular viscoelastic device (OVD) in order to proceedwith the removal of the anterior capsule paying attention toany tissue capsular bridges to avoid capsule fugue

Hydrodissection and hydrodelineation were performedusing a 26-gauge cannula with injection of BSS under theanterior capsule edge paying attention to the air bubblesmovements in order to avoid any posterior capsular blockand ruptures Bimanual phacoemulsification technique wasused to aspirate the nucleus with a 20-gauge 30-degree angledsleeveless probe and a 19-gauge irrigating chopper (Oertli-Instruments AG) Finally bimanual irrigationaspiration ofthe residual cortex was performed using two 21-gauge probeswith an oval-shaped section (21-gauge irrigation handpiecesmooth 21-gauge aspiration handpiece rough Bausch ampLomb) followed by posterior capsule polishing All intraoc-ular lenses were placed in the capsular bag without compli-cations Anterior lens capsule removal after either manualanterior capsulorhexis or FSL-assisted anterior capsulotomyis shown in Figure 1

212 Standard Technique The surgeon performed two14mm biplanar trapezoidal incisions at 10 orsquoclock and 2orsquoclock respectively with a precalibrated knife and a man-ual 5mm continuous curvilinear capsulorhexis (mCCC)Hydrodissection was performed with 26-gauge cannulaand phacoemulsification with a 20-gauge 30-degree angledsleeveless probe and an irrigating chopper (Bragamele 19-gauge) Phaco-fracture was obtained by the stop-and-chop

BioMed Research International 3

(a) (b)

Figure 1 Anterior lens capsule removal in B-MICS and FLACS surgical techniques (a) Manual anterior capsulorhexis with micro-forceps (b)appearance of the lens after FLACS and anterior capsulotomy removal with micro-forceps

technique Irrigationaspiration was performed with two 21-gauge probeswith an oval-shaped section (21-gauge irrigationhandpiece smooth Stellaris 21-gauge aspiration handpiecerough Bausch amp Lomb) In both techniques IOL wasimplanted without enlargement of the main incision theincisions were hydrosutured BunnyLens AF IOL (HanitaLenses Israel) were implanted through 14mmmicroincisionwith the wound assisted technique through the ViscoJectBIO 15 injector At the end of surgery OVD was removedand the incisions were hydrated for safety reasons

Postoperative therapy consisted in tobramycin and dex-amethasone eye drops four times a day for 15 days followedby flurbiprofen eye drops three times a day for further 15 days

22 Morphology Evaluation Phase Contrast Histological andScanning ElectronMicroscopyAnalyses Anterior lens capsulespecimens obtained from either FSL-assisted or standardtechniques were immediately washed with phosphate buffersaline (PBS) following surgical extraction In order to evalu-ate the morphology and the edges of the capsules obtainedfollowing both the cataract surgical techniques specimenswere processed for phase contrast histological and scanningelectron microscopy analyses Part of the samples were fixedwith 4 paraformaldehyde (PFA) in PBS for 20 minutes atroom temperature Then samples were washed thrice withPBS and were observed by using a Nikon Eclipse TE2000-U invertedmicroscope Representative phase contrast imageswere acquired with a CCD Hamamatsu ORCA 285 cameraAfterwards samples were processed for histological analysisby undergoing dehydrationwith graded ethanol clearing andembedding in paraffin Five 120583m thick serial sections werecut for each sample and routine haematoxylineosin (HampE)stainingwas performed in order to analyze themorphologicaldetails and measure the thickness of the anterior capsules

Another part of the collected samples was washed rightafter surgical procedures with 01M pH 74 Sorensonrsquos Phos-phate Buffer (PB) and fixed with 1 glutaraldehyde in 01MpH 74 PB After three washings in PB anterior lens capsulesamples were dehydrated with graded ethanol until SEMobservation was performed SEM images were acquired with

a Nova NanoSEM 450 (FEI) scanning electron microscope(SEM)

The main aim of these analyses was the evaluation of theepithelial cells layer the presence of fringes in the capsuleedges and the roughnesssmoothness of the edges

23 Evaluation of Apoptosis in LECs In order to evaluatewhether apoptosis occurred in lens epithelial cells adheringto anterior lens capsule following B-MICS surgical approachand after release of pulse energy by femtosecond lasersamples were processed for confocal immunofluorescenceanalysis as previously described [13 14] Briefly capsulesfixed with 4 were washed with PBS and underwent sat-uration with PBS containing 3 bovine serum albumin(BSA) for 30 minutes at room temperature Then sampleswere incubated with a rabbit anti-cleaved caspase 3 primaryantibody (Cell Signaling) diluted 1 50 in PBS containing 3BSA for 1 hour at room temperature After washing in PBScontaining 3 BSA specimens were incubated with a goatanti-rabbit Alexa546 secondary antibody (Life Technologies)diluted 1 200 in PBS containing 3 BSA Samples were thenwashed with PBS stained with 1mgml 410158406-diamidino-2-phenylindole (DAPI) in PBS for 1 minute and then mountedwith antifading medium Fluorescent samples were observedby a Nikon A1 confocal laser scanning microscope Theconfocal serial sections were processed with ImageJ softwareto obtain three-dimensional projections and image renderingwas performed using Adobe Photoshop Software [15]

24 Statistical Analysis Data were evaluated by statisticalsoftware (GraphPad Prism Software version 7 Inc SanDiegoCA USA) Differences between the two experimental groupswere analyzed by Studentrsquos 119905-test Data were expressed asmean plusmn SD Each experimental evaluation was performed intriplicate In any case significance was set at 119875 lt 005

3 Results

31 Clinical Observations For Group 1 (FLACS) 30 eyes (15right eyes and 15 left eyes) of 30 patients (10 males and 20


(a) (b)

Figure 2 Histomorphology evaluation of anterior lens capsules Phase contrast images showing edges and epithelial layer of anterior lenscapsules obtained from B-MICS technique (a) and femtosecond laser assisted cataract surgery (b) Bar 100120583m

females) were included in the study the average age was 7407plusmn 848 years For Group 2 (B-MICS) 30 eyes (13 right eyesand 17 left eyes) of 30 patients (8 males and 22 females) wereincluded in the study the average age was 7572 plusmn 916 yearsBunnyLens AF IOL was implanted in all eyes All IOLs wereimplanted in the bag

The total surgical time was 1789 plusmn 380 minutes in Group1 whereas in Group 2 it was 1263 plusmn 210 minutes with adifference resulting to be statistically significant (119875 lt 005)In Group 1 no incomplete capsulotomy nor capsule buttonswere observed moreover no intraoperative major complica-tion nor any postoperative complicationwas recorded in bothgroups

Three months after surgery a mean BCVA improvementof 0460 plusmn 0249 LogMar was observed in Group 1 (119875 lt 005versus baseline values) Similarly in Group 2 a mean BCVAimprovement of 0417 plusmn 0150 LogMar (119875 lt 005 versusbaseline values) was observed at the same evaluation timeNo statistically significant differences in the improvement ofvisual acuity were observed between the two groups

With regard to astigmatism and corneal pachymetry wedid not detect any statistically significant difference betweenbaseline and postoperative values with Studentrsquos 119905-test eitherwithin each group or between the two groups (119875 gt 005)

Finally with regard to the endothelial cell count (ECC) inGroup 1 at 3-month follow-up we found a mean endothelialcell loss of 16227 plusmn 22511 cellsmm2 and of 32252 plusmn 33993cellsmm2 after surgery in Group 2 a reduction that wasstatistically significant either within each group or betweenthe two groups (119875 lt 005)

32 Morphological Analysis Phase Contrast Histologicaland Scanning Electron Microscopy Analyses Phase contrastimages showed epithelial cells with well-conserved nucleiand cytoplasm besides revealing intact cellular bordersthrough most of the epithelial surface except for the closerproximity of the capsular edges where epithelial cells weredetached from the basement membrane (Figure 2(a)) Edgesof the capsules appeared regular and smooth as reportedin Figure 2(a) As far as the capsular appearance in FLACSgroup is concerned epithelial cells were mostly adherent

B-M

ICS

grou

pFL

ACS

gro

up

Figure 3 Histological analysis of anterior lens capsules Crosssections of the specimenswere stainedwith haematoxylin and eosinOn the right side higher magnification images show details of theepithelial layer in specimens obtained followingB-MICS andFLACStechniques Bar 10 120583m

to the basement membrane whereas edges showed a lessregular saw-tooth pattern (Figure 2(b)) Histological analysisconfirmed that some of the samples from the B-MICS groupshowed a partial detachment of the epithelial layer from thebasement membrane whereas most of the capsules fromthe FLACS group demonstrated a continuous adhesion ofepithelial cells and basement membrane (Figure 3) Further-moremeasurements of the capsular thickness among the twoexperimental groups revealed a difference although withoutstatistical significance In particular samples obtained afterFLACS technique showed a higher thickness when comparedto capsules derived from B-MICS (data not shown)

Finally SEM analysis as reported in Figure 4 revealedwith a higher definition how uniform the adhesion ofepithelial cells to the basement membrane was and most ofall the appearance of capsular edges In particular in theB-MICS group the capsular edges had a regular smoothand translucent rim (Figure 4(a)) Otherwise the margins


1000x 4000x2000x

(a)

B

1000x2000x 4000x

(b)

Figure 4 Scanning electron microscopy analysis Representative images of samples obtained with B-MICS (a) and (b) FLACS techniquesshowing epithelial layer (left) and edges appearance (right) at different magnifications

DAPI cleaved casp3

DAPI cleaved casp3

B-M

ICS

grou

pFL

ACS

gro

up

(a)B-MICS Femto

0

2

4

6

8

Casp

ase 3

(int

egra

ted

dens

ity) lowastlowast

(b)

Figure 5 Evaluation of cell apoptosis in epithelial cells adherent to anterior lens capsules (a) Immunofluorescence analysis was performedagainst cleaved caspase 3 (red) and nuclei were counterstained with DAPI (blue) (b) Pseudocolor images (upper) and densitometric analysis(lower) of cleaved caspase 3 in epithelial cells Bar 20120583m

of capsules obtained through FLACS clearly demonstratedirregular and fringed appearance (Figure 4(b))

33 Evaluation of Apoptosis in LECs Confocal immunoflu-orescence analysis revealed that apoptosis occurred in lens

epithelial cells of samples obtained from either the B-MICSgroup or the FLACS group as shown in Figure 5(a) Whilein capsules obtained from the manual surgical techniquethe presence of cleaved caspase 3 was observed mainly inepithelial cells closer to the capsular edges samples collected


following FLACS technique showed a more spread stainingagainst cleaved caspase 3 from the rim to the core of theepithelial layer Pseudocolor images of cleaved caspase 3were obtained as previously described [16] and reportedin Figure 5(b) Densitometric analysis carried out on theimmunofluorescent signals provided semiquantitative dataof the expression of the apoptotic effector cleaved caspase3 which was statistically significantly higher in the FLACSgroup with respect to the B-MICS group (Figure 5(b) lowastlowast119875 lt001)

4 Discussion

Theuse of femtosecond lasers constitutes the newest achieve-ment in cataract surgery starting from their first clinicalapplication in 2008Due to the increasing necessity to addressthe expectations of optimum refractive outcome a lot of newdevelopments have been fulfilled

The application of femtosecond laser to cataract surgeryhas allowed improving the safety and effectiveness of theprocedure as well documented in literature [5 7 9 17]Indeed previous reports demonstrated the high repeatabilityand precision in execution of the most difficult phases ofcataract surgery including optimized anterior capsulotomieswhich produced a continuous sharp-edged cut with goodtensile strength [9 18 19]

Our study evaluated themorphological features of humananterior capsules obtained with standard phacoemulsifica-tion technique and FLACS respectively The latter ones werecollected following the use of Ziemer LDVZ8 a femtosecondlaser that takes advantage of a low energy high frequencysystem (high pulse repetition ratemdashup to 10MHzmdashcombinedwith a very low energy range of 100ndash150 nJ) [20] and usesa liquid optic interface to ease the capsulotomy and lensfragmentation phases These properties allowed obtaininganterior capsule specimens owning a well-preserved histo-morphology with epithelial cells uniformly adherent whencompared to samples obtained after manual technique asdemonstrated by phase contrast and SEM analyses In accor-dance with the existing literature the edges of anterior cap-sules derived fromFLACSwere less regular and characterizedby a saw-tooth pattern when compared to the smooth rimproduced bymanual technique [21 22]The pattern observedin the FLACS group is most likely attributable to high densitypulse raster and high precision optics of the Femto LDV Z8laser which result in multiple overlapping spots that createa continuous clean cut resembling the one obtained with B-MICS technique [23]

Anterior capsule specimens obtained through FLACStechnique also showed a higher thickness when comparedto capsules derived from B-MICS most likely due to thepresence of cortex adherent to the anterior capsule

Besides histomorphological characteristics our datashowed that apoptosis occurred in epithelial cells of anteriorcapsules obtained from both surgical techniques In particu-lar cleaved caspase 3 expression was significantly higher insamples collected following FLACS with a positive stainingnot limited to the cells along the cutting edge but also spreadto the inner core of the capsule thus suggesting a wider

induction of cell death Conversely in the epithelial layeradhering to the capsule specimens derived from B-MICSprocedure apoptosis was detected only along the cuttingrim as a consequence of mechanical injury induced bythe forceps mediated extraction of anterior capsule Thisgreater apoptotic event affecting the monolayered epitheliumbeneath the anterior capsule after FLACS is likely due to laserpulse energy photodisruption-dependent plasma formationand subsequent creation of cavitation bubbles [4] Our dataare consistent with previous findings from Mayer et al [24]and particularly a spread expression of the apoptotic effectorcleaved caspase 3 might represent a positive predicting factorthat allows preventing capsular bag opacification by stronglyinhibiting the proliferation of residual epithelial cells whichgenerally correlates to postoperative inflammatory response[25 26] From a clinical point of view our study highlightedthat 3months after surgery patients from either FLACS or B-MICS groups showed a statistically significant improvementin best-corrected visual acuity (BCVA) values with respect tothe baseline measurements Noteworthy as far as endothelialcell count is concerned a statistically significant decrease incorneal endothelial cell loss was observed in FLACS groupevent that is attributable to shorter phacoemulsificationtimes These data are in accordance with previous findingsdemonstrating that laser cataract surgery is less traumatic tothe corneal endothelium besides offering a tool for surgeonsto standardize the critical phases of cataract surgery throughan initial learning curve [27ndash29]

5 Conclusions

The use of femtosecond laser in cataract surgery allowsoptimizing some of the most difficult steps of the procedureincluding capsulotomy nuclear fragmentation and cornealincisions besides providing more accurate and repeatableoutcomes Our findings are in accordance with the existingliterature however further studies will be needed to matchthe bright sides of manual technique and femtosecond laserby defining the optimal combination of laser energy repe-tition rate and spot size in order to obtain better qualitycapsulotomieswith regular cutting edges comparable to thoseof manual capsulorhexis and to reduce the inflammatoryresponse in the eye thus preventing lens epithelial cellproliferation and capsular bag opacification

Abbreviations

FLACS Femtosecond assisted laser cataract surgeryB-MICS Bimanual microincision cataract surgerymCCC Manual continuous curvilinear capsulorhexisIOL Intraocular lensPCO Posterior capsule opacificationBSS Balanced salt solutionOCT Optical coherence tomographyOVD Ocular viscoelastic devicePBS Phosphate buffer salinePFA ParaformaldehydeHampE Haematoxylineosin


SEM Scanning electron microscopeBSA Bovine serum albuminDAPI 410158406-Diamidino-2-phenylindole

Disclosure

This work was presented in part at the 20th AICCERmeeting2017 (Associazione Italiana di Chirurgia della Cataratta eRefrattiva) March 9ndash11 Rimini (Italy) 2017

Conflicts of Interest

The authors declare that no financial conflicts of interestexist

References

[1] S Koopman Cataract Surgery Devices ndash Global Pipeline Analy-sis Competitive Landscape andMarket Forecasts to 2017 Global-Data London UK 2012

[2] G M Cavallini L Campi C Masini S Pelloni and A PupinoldquoBimanual microphacoemulsification versus coaxial minipha-coemulsification Prospective studyrdquo Journal of Cataract ampRefractive Surgery vol 33 no 3 pp 387ndash392 2007

[3] GM Cavallini T VerdinaM Forlini et al ldquoLong-term follow-up for bimanual microincision cataract surgery Comparisonof results obtained by surgeons in training and experiencedsurgeonsrdquo Clinical Ophthalmology vol 10 pp 979ndash987 2016

[4] Z Z Nagy ldquoNew technology update femtosecond laser incataract surgeryrdquo Clinical Ophthalmology vol 8 pp 1157ndash11672014

[5] S J Bali C Hodge M Lawless T V Roberts and G SuttonldquoEarly experience with the femtosecond laser for cataractsurgeryrdquo Ophthalmology vol 119 no 5 pp 891ndash899 2012

[6] T V Roberts M Lawless C C Chan et al ldquoFemtosecond lasercataract surgery technology and clinical practicerdquo Clinical ampExperimental Ophthalmology vol 41 pp 180ndash186 2012

[7] L He K Sheehy and W Culbertson ldquoFemtosecond laser-assisted cataract surgeryrdquo Current Opinion in Ophthalmologyvol 22 pp 43ndash52 2011

[8] MBaumeister BNeidhardt J Strobel andTKohnen ldquoTilt anddecentration of three-piece foldable high-refractive silicone andhydrophobic acrylic intraocular lenses with 6-mm optics in anintraindividual comparisonrdquo American Journal of Ophthalmol-ogy vol 140 no 6 pp 1051ndash1058 2005

[9] N J Friedman D V Palanker G Schuele et al ldquoFemtosecondlaser capsulotomyrdquo Journal of CataractampRefractive Surgery vol37 no 7 pp 1189ndash1198 2011

[10] T Chan U Pattamatta M Butlin K Meades and C BalaldquoIntereye comparison of femtosecond laser-assisted cataractsurgery capsulotomy and manual capsulorhexis edge strengthrdquoJournal of Cataract amp Refractive Surgery vol 43 no 4 pp 480ndash485 2017

[11] R Svancarova J Novak and H Adamkova ldquoThe influence oflens capsule mechanical polishing to the secondary cataractdevelopmentrdquo Ceska a Slovenska Oftalmologie vol 66 no 1 pp21ndash25 2010

[12] N Awasthi S Guo and B J Wagner ldquoPosterior capsularopacification a problem reduced but not yet eradicatedrdquo JAMAOphtalmology vol 127 no 4 pp 555ndash562 2009

[13] A Pisciotta M Riccio G Carnevale et al ldquoStem cells isolatedfrom human dental pulp and amniotic fluid improve skeletalmuscle histopathology in mdxSCID micerdquo Stem Cell ResearchampTherapy vol 6 no 1 article 156 2015

[14] G Carnevale G Carpino V Cardinale et al ldquoActivation ofFasFasL pathway and the role of c-FLIP in primary culture ofhuman cholangiocarcinoma cellsrdquo Scientific Reports vol 7 no1 2017

[15] A Pisciotta M Riccio G Carnevale et al ldquoHuman serumpromotes osteogenic differentiation of human dental pulp stemcells in vitro and in vivordquo PLoS ONE vol 7 no 11 Article IDe50542 2012

[16] M Riccio G Carnevale V Cardinale et al ldquoThe FasFas ligandapoptosis pathway underlies immunomodulatory properties ofhumanbiliary tree stemprogenitor cellsrdquo Journal ofHepatologyvol 61 no 5 pp 1097ndash1105 2014

[17] R Naranjo-Tackman ldquoHow a femtosecond laser increasessafety and precision in cataract surgeryrdquo Current Opinion inOphthalmology vol 22 no 1 pp 53ndash57 2011

[18] Z Nagy A Takacs T Filkorn and M Sarayba ldquoInitial clinicalevaluation of an intraocular femtosecond laser in cataractsurgeryrdquo Journal of Refractive Surgery vol 25 no 12 pp 1053ndash1060 2009

[19] L Mastropasqua L Toto R Calienno et al ldquoScanning electronmicroscopy assisted cataract surgeryrdquo Journal of Cataract ampRefractive Surgery vol 39 no 10 pp 1581ndash1586 2013

[20] G P Williams B L George Y R Wong et al ldquoThe effects of alow-energy high frequency liquid optic interface femtosecondlaser system on lens capsulotomyrdquo Scientific Reports vol 6Article ID 24352 2016

[21] S Nowroozizadeh N Cirineo N Amini et al ldquoInfluence ofcorrection of ocular magnification on spectral-domain OCTretinal nerve fiber layer measurement variability and perfor-mancerdquo Investigative Ophthalmology amp Visual Science vol 55no 6 pp 3439ndash3446 2014

[22] S Serrao G Lombardo G Desiderio et al ldquoAnalysis of fem-tosecond laser assisted capsulotomy cutting edges and manualcapsulorhexis using environmental scanning electron micro-scopurdquo Journal of Ophthalmology Article ID 520713 2014

[23] M Tomita A Chiba J Matsuda and Y Nawa ldquoEvaluation ofLASIK treatment with the femto LDV in patients with cornealopacityrdquo Journal of Refractive Surgery vol 28 no 1 pp 25ndash302012

[24] W J Mayer O K Klaproth M Ostovic et al ldquoCell death andultrastructural morphology of femtosecond laser-assisted ante-rior capsulotomyrdquo Investigative Ophthalmology amp Visual Sci-ence vol 55 no 2 pp 893ndash898 2014

[25] E Bertelmann and C Kojetinsky ldquoPosterior capsule opacifi-cation and anterior capsule opacificationrdquo Current Opinion inOphthalmology vol 12 no 1 pp 35ndash40 2001

[26] J-M Rakic A Galand and G F J M Vrensen ldquoLens epithelialcell proliferation in human posterior capsule opacificationspecimensrdquo Experimental Eye Research vol 71 no 5 pp 489ndash494 2000

[27] A I Takacs I Kovacs K Mihaltz T Filkorn M C Knorz andZ Z Nagy ldquoCentral corneal volume and endothelial cell countfollowing femtosecond laser-assisted refractive cataract surgerycompared to conventional phacoemulsificationrdquo Journal ofRefractive Surgery vol 28 no 6 pp 387ndash391 2012

[28] I Conrad-Hengerer M Al Juburi T Schultz F H Hengererand H B Dick ldquoCorneal endothelial cell loss and corneal


thickness in conventional compared with femtosecond laser-assisted cataract surgery Three-month follow-uprdquo Journal ofCataract amp Refractive Surgery vol 39 no 9 pp 1307ndash1313 2013

[29] G M Cavallini T Verdina M De Maria E Fornasari EVolpini and L Campi ldquoFemtosecond laser-assisted cataractsurgery with bimanual technique learning curve for an expe-rienced cataract surgeonrdquo International Ophthalmology 2017

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fragmentation and corneal incisions besides providingmoreaccurate and reproducible results when compared to manualapproaches [4]

Until the advent of femtosecond laser technology manualcontinuous curvilinear capsulorhexis (mCCC) has been thestandard method for the opening of anterior capsule incataract surgery or refractive lens replacement Femtosecondlaser assisted cataract surgery (FLACS) has now revolution-ized this important surgical step creating predictable andcurvilinear capsulotomies which result in improved capsule-intraocular lens (IOL) overlap and better IOL centration andposition than with the manual technique In particular pre-vious findings demonstrated that FLACS capsulotomies havea repeatable and precise size are centered and consequentlyprovide better IOL centration and reduced probability of IOLtilt [5ndash9]

Several studies have examined the morphology of theedges and the smoothness of FLACS capsulotomies whichare created by specific laser wavelength pulse energy pulseduration pulse pattern repetition rate and spot size [4]Preliminary analyses of the capsules showed a postage-stamp aspect with a significant roughness likely caused byseveral factors including the laser energy used to create thecapsulotomies [10]

Moreover posterior capsule opacification (PCO) is oneof the most recurring long-term complications of cataractsurgery Many studies have been conducted so far in orderto define which factors may influence the developmentof PCO including the IOL design the administration oftherapeutic agents the surgical technique itself and othersurgical procedures to clean the posterior capsule [11 12]

As reported in the literature lens capsule is exposed tomechanical or photodisruptive damage when either capsu-lorhexis or capsulotomy is performed with consequent deathof the epithelial cellsThe hypothesis is that femtosecond lasermight reasonably induce apoptosis to a higher extent

The aim of this study was to evaluate morphologicalfeatures of the human anterior lens capsule even at theultrastructural level following cataract surgery performedthrough manual technique and FLACS respectively Fur-thermore we investigated whether apoptosis was inducedin monolayered epithelium beneath the anterior capsulefollowing the two surgical approaches

2 Materials and Methods

The experimental study was approved by the Human Re-search Ethics Committee of the University of Modena andReggio Emilia (Modena Italy) and was conducted in com-pliance with the Declaration of Helsinki at the Institute ofOphthalmology University of Modena and Reggio EmiliaPatients with cataract LOCS III grade 2 or 3 and a correctdistance visual acuity of 510 or worse were included inthe study Patients were excluded in case of ocular traumapseudoexfoliation syndrome or other ocular comorbiditiesThe study was performed after written consent of the patientsundergoing either traditional manual cataract extraction (B-MICS) or femtosecond laser assisted cataract surgery byusing the femtosecond laser Ziemer LDV Z8 All the surgical

procedures were performed by the same experienced surgeon(GMC)

21 Surgical Techniques A consistent mydriasis was obtainedwith instillation of 10 phenylephrine and 1 cyclopentolateand a locoregional anaesthesia with peribulbar block wasperformed (15mL of lidocaine 2 and 15mL of bupivacaine05)

211 FSL-Assisted Technique We carefully applied a dispos-able suction ring to the eye centered over the limbus Thesuction ring was filled with balanced salt solution (BSS)in order to create a liquid interface and the mobile armof the laser system was docked over the corneal apexThe ocular structures were shown by the integrated opti-cal coherence tomography (OCT) system and treatmentparameters were determined in a customized manner byusing the laser platform settings wizard The energy andfrequency laser pulse settings were 900mW 1MHz forcapsulotomy 950ndash1000mW 2MHz for phacofragmentation1200ndash1300mW 2MHz for corneal incisions Laser treatmentstarted with lens fragmentation with an eight-piece pie-cut pattern followed by an anterior capsulotomy of 52mmdiameter Finally the laser performed two 14mm cornealmicroincisions at 10 orsquoclock and 2 orsquoclock respectively Atthe end the suction ring was removed from the eye surfaceto proceed with the phaco procedure The surgeon checkedthe patency of the CCIs using a smooth spatula first with aperpendicular inclination to the limbus inclination and thenwith gentle movements along the incision in order to open itcompletely from side to side The anterior chamber was filledwith ocular viscoelastic device (OVD) in order to proceedwith the removal of the anterior capsule paying attention toany tissue capsular bridges to avoid capsule fugue

Hydrodissection and hydrodelineation were performedusing a 26-gauge cannula with injection of BSS under theanterior capsule edge paying attention to the air bubblesmovements in order to avoid any posterior capsular blockand ruptures Bimanual phacoemulsification technique wasused to aspirate the nucleus with a 20-gauge 30-degree angledsleeveless probe and a 19-gauge irrigating chopper (Oertli-Instruments AG) Finally bimanual irrigationaspiration ofthe residual cortex was performed using two 21-gauge probeswith an oval-shaped section (21-gauge irrigation handpiecesmooth 21-gauge aspiration handpiece rough Bausch ampLomb) followed by posterior capsule polishing All intraoc-ular lenses were placed in the capsular bag without compli-cations Anterior lens capsule removal after either manualanterior capsulorhexis or FSL-assisted anterior capsulotomyis shown in Figure 1

212 Standard Technique The surgeon performed two14mm biplanar trapezoidal incisions at 10 orsquoclock and 2orsquoclock respectively with a precalibrated knife and a man-ual 5mm continuous curvilinear capsulorhexis (mCCC)Hydrodissection was performed with 26-gauge cannulaand phacoemulsification with a 20-gauge 30-degree angledsleeveless probe and an irrigating chopper (Bragamele 19-gauge) Phaco-fracture was obtained by the stop-and-chop


(a) (b)










3 Results



(a) (b)








B-M

ICS

grou

pFL

ACS

gro

up





1000x 4000x2000x

(a)

B

1000x2000x 4000x

(b)


DAPI cleaved casp3

DAPI cleaved casp3

B-M

ICS

grou

pFL

ACS

gro

up

(a)B-MICS Femto

0

2

4

6

8

Casp

ase 3

(int

egra

ted

dens

ity) lowastlowast

(b)







4 Discussion







5 Conclusions


Abbreviations




Disclosure




References




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a) (b)










3 Results



(a) (b)








B-M

ICS

grou

pFL

ACS

gro

up





1000x 4000x2000x

(a)

B

1000x2000x 4000x

(b)


DAPI cleaved casp3

DAPI cleaved casp3

B-M

ICS

grou

pFL

ACS

gro

up

(a)B-MICS Femto

0

2

4

6

8

Casp

ase 3

(int

egra

ted

dens

ity) lowastlowast

(b)







4 Discussion







5 Conclusions


Abbreviations




Disclosure




References




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a) (b)








B-M

ICS

grou

pFL

ACS

gro

up





1000x 4000x2000x

(a)

B

1000x2000x 4000x

(b)


DAPI cleaved casp3

DAPI cleaved casp3

B-M

ICS

grou

pFL

ACS

gro

up

(a)B-MICS Femto

0

2

4

6

8

Casp

ase 3

(int

egra

ted

dens

ity) lowastlowast

(b)







4 Discussion







5 Conclusions


Abbreviations




Disclosure




References




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















1000x 4000x2000x

(a)

B

1000x2000x 4000x

(b)


DAPI cleaved casp3

DAPI cleaved casp3

B-M

ICS

grou

pFL

ACS

gro

up

(a)B-MICS Femto

0

2

4

6

8

Casp

ase 3

(int

egra

ted

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ity) lowastlowast

(b)







4 Discussion







5 Conclusions


Abbreviations




Disclosure




References




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















4 Discussion







5 Conclusions


Abbreviations




Disclosure




References




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















Disclosure




References




































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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