RETINAL DISORDERS

The novel use of decorin in prevention of the developmentof proliferative vitreoretinopathy (PVR)

Khaled Nassar & Julia Lüke & Matthias Lüke & Mahmoud Kamal & Effat Abd El-Nabi &Mahmoud Soliman & Martin Rohrbach & Salvatore Grisanti

Received: 21 February 2011 /Revised: 21 April 2011 /Accepted: 24 April 2011 /Published online: 7 July 2011# Springer-Verlag 2011

AbstractBackground The cytokine transforming growth factor-ß(TGF-ß) is a pivotal contributor to tissue fibrosis and akey cytokine in the pathogenesis of cellular transdiffer-entiation, epithelial-mesenchymal transition (EMT), andcell adhesion. This study evaluates the effect of decorin, anaturally occurring TGF-ß inhibitor, in an experimentalrabbit model for proliferative vitreoretinopathy (PVR).Methods Traumatic PVR was induced in 50 rabbits dividedinto ten groups (n=5). One group (GI) reveals a controlwith no treatment after trauma. Groups (GII–GIV) con-sisted of subgroups receiving phacovitrectomy at threedifferent time points; (a) at the time of trauma, (b) 1 week

following trauma, and (c) 2 weeks following trauma. GIIIand GIV received 100 μg or 200 μg decorin, respectively.PVR severity was scored from 0 to 4. The amount offibrosis was quantified using JMicroVision© software.Results The control group GI developed severe PVR withtractional retinal detachment (TRD); (PVR score ≥2) in fourrabbits out of five. Vitrectomy had a positive effect (p<0.05) on PVR development when preformed immediately,however the developed fibrosis was high. The best resultswere obtained when surgery was used in conjunction withdecorin that reduced both the PVR score and fibrosisdevelopment significantly (p

extracellular matrix (ECM) proteoglycans, known as smallleucine-rich proteoglycans (SLRPs) [3]. It is composed of a40-kDa protein core and one chondroitin/dermatan sulfateside chain. Decorin plays a key role in the regulation ofECM assembly by binding to several components such ascollagen [4–6], thrombospondin [7], and fibronectin [8, 9].Interaction of decorin with collagen has been shown toaffect fibril formation by causing an initial delay in thelateral assembly and a reduction of the average fibrildiameter [4, 10]. In addition to its ability to modulate theECM assembly, decorin also displays a number of biologiceffects through binding to growth factors such as trans-forming growth factor beta (TGF-ß) [11, 12] and withconnective tissue growth factor (CTGF) [13]. Furthermore,it has been demonstrated that decorin has an inhibitoryeffect on platelet-derived growth factor (PDGF), anothermajor player in wound healing [14]. Decorin is alsoinvolved in the control of cell growth by binding to theepidermal growth factor (EGF) receptor [15], leading toactivation of mitogen-activated protein (MAP) kinases andinduction of the growth suppressor p21 [15–17]. Inaddition, decorin regulates intralysosomal degradation [18].

Proliferative vitreoretinopathy (PVR) is characterized bythe formation of fibrotic membranes within the vitreous andat the retina. Contraction of these membranes may causevitreoretinal traction, traction-related retinal breaks, recur-rent retinal detachment (RD), and ocular hypotony [19]. Avariety of studies indicate that PVR is a complex processcomprising events that are similar to those of the woundhealing response with inflammation, migration, and prolif-eration of a variety of cells [20, 21]. Different studiessuggest that retinal pigment epithelial cells (RPE) contrib-ute to fibrosis by epithelial-mesenchymal transition (EMT)[22, 23]. PVR developed following RD showed detachmentof RPE from their normal location. Then they either migrateor are swept in fluid vitreous to the retinal surfaces, wherethey undergo EMT to form several morphologic typesincluding fibroblast-like cells [22]. TGF-ß is a pivotalcontributor to tissue fibrosis and a key cytokine in the

pathogenesis of EMT [23]. TGF-ß levels are elevated inPVR vitreous and they correlate with the presence ofintraocular fibrosis [24]. Neutralizing TGF-ß antibodieshave been shown to reduce experimental dermal scarring[25] and have also been introduced into glaucoma filtrationsurgery [26, 27]. Grisanti et al. reported the potential use ofdecorin [28] in glaucoma filtering surgery without evidentocular toxicity. Inspired by these studies, we wanted toanalyze whether decorin could be used instead of thepolyclonal antibodies [25] or the more demanding recom-binant human anti-TGF-ß antibodies [27] to prevent post-traumatic PVR.

The current study evaluated the use of decorin as anadjuvant anti-fibrotic treatment in a rabbit model of PVR[29]. The rabbit model was preferred over a primate model[30] because it is well established, cost-effective, and itsaggressive wound-healing response makes it equivalent tohigh-risk eyes in humans [31]. The model involves oculartrauma and vitreous hemorrhage induction. Both of themare important risk factors for PVR development [32, 33].

Materials and methods

Animals

All experiments were performed with female chinchillabastard rabbits (Crl:CHB) 3–6 months old and weighing1.5–2.5 kg. The animals were obtained from Charles RiverLaboratories (Sulzfeld, Germany) and acclimatized for1 week before the experiments started. Traumatic PVRwas induced in 50 rabbits equally divided into ten groups.One group (GI) served as the control and the other groupsdiffered in the time of surgical intervention (phacovitrec-tomy) and dosage of decorin (100 μg and 200 μg) that wasintravitreally injected (Table 1). Principles of LaboratoryAnimal Care (NIH publication No. 85–23, revised 1985),the OPRR Public Health Service Policy on the HumaneCare and Use of Laboratory Animals (revised 1986), and

Group Subgroup na Trauma Vitrectomy time in relation to trauma IVTb decorin dose

GI - 5 Trauma - -

GII GIIa 5 Trauma Immediate -GIIb 5 Trauma 1 week

GIIc 5 Trauma 2 weeks

GIII GIIIa 5 Trauma Immediate 100 μg /0.1 mlGIIIb 5 Trauma 1 week

GIIIc 5 Trauma 2 weeks

GIV GIVa 5 Trauma Immediate 200 μg/0.1 mlGIVb 5 Trauma 1 week

GIVc 5 Trauma 2 weeks

Table 1 Animal groups

na number of rabbits

IVTb intravitreal

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the U.S. Animal Welfare Act, as amended, were followed,as well as the current version of the German Law on theProtection of Animals were applied.

Anesthesia

For anesthesia and sedation, ketamine (Ketanest; Parke Davis,Berlin, Germany), xylazine (Rompun; Bayer, Leverkusen,Germany) and local anesthesia with oxybuprocaine drops(Novesine 0.4%; Novartis, Nürnberg, Germany) were used.General anesthesia was achieved with intramuscular (IM)injection of 25 mg/kg-body weight ketamine and 2 mg/kg-body weight xylazine, and after ½ hour, another half dosewas given. Sedation was achieved with intramuscularinjection of 12.5 mg/kg-body weight ketamine and 1mg/kg-body weight xylazine, and after ½ hour, another halfdose was given.

Surgical procedure

Due to the impact of the procedure on the visual abilityof the animal, only the right eyes were treated. Surgerywas performed under general anesthesia as describedbefore. All surgeries were done by the same surgeon.Briefly, the pupils were maximally dilated. Preoperativefundus examination was done to exclude the presence ofa pre-existing fundus abnormality. Principles of aseptictechnique and preoperative care were applied and surgerywas done with the use of an operating microscope (Zeiss

OPMI, Jena, Germany). In all groups, a previouslydescribed rabbit model of traumatic PVR was used[29]. We have done two modifications for this model.First, we used a specifically designed incision marker tostandardize the trauma induction site. Second, we used amixture of 1% buffered formaldehyde and 1.25% glutar-aldehyde as a fixation solution for the enucleated eyes.Briefly, while the specifically designed incision markerwas held in place, an 8-mm oblique scleral incision wascarried out at a distance of 1 mm and 2 mm behind thelimbus. The incision was performed at the upper nasalquadrant. The wound was sutured with three interrupted8–0 Vicryl sutures (Ethicon, Johnson and Johnson Intl,Belgium) and 0.4 ml autologous blood was injectedintravitreally (Fig. 1a). In GI, only this procedure wasdone. In GII, GIII, and GIV; pars plana vitrectomy wasdone using a vitrectomy machine (Storz, Bausch andLomb, Berlin, Germany). Vitrectomy was either preformedimmediately after trauma and wound closure (subgroupsa), or 1 week (subgroups b) or 2 weeks (subgroups c)thereafter (Table 1). Phacovitrectomy technique wasmodified to adapt to the rabbit’s ocular anatomy. A lateralcanthotomy was preformed followed by eyelid speculumapplication. The lens was then emulsified and aspiratedthrough corneal approach and the corneal wounds weresecurely sutured. Then two preplaced limbal sutures wereused to fix a vitrectomy contact lens ring holding a contactvitrectomy lens. Pars plana vitrectomy was then preformedusing one-step 23-gauge vitrectomy system (D.O.R.C.

Fig. 1 Surgical procedure. aInduction of traumatic PVR:While the marker (arrow) washeld in place, an oblique scleralincision was made. Then thewound was repaired followed byintravitreal injection of 0.4 ml ofautologous blood. b Vitrectomy:Core vitrectomy was done forremoval of the vitreous hemor-rhage (arrow) and the developedmembranes. c Posterior vitreousdetachment (PVD): PVD wasinduced using the vitrectomycutter. While the suction-onlymode of the foot pedal wasapplied, the posterior vitreous(arrow) was entangled andcarefully detached from the ret-inal surface. d Vitreous baseremoval: the retinal peripherywas indented and the vitreousbase (arrow) was removed. Theretina was examined for thepresence of tears or detachment

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Deutschland GmbH, Berlin, Germany). Sclerotomies werefashioned 1.5 mm from the limbus. Vitrectomy wasperformed at high cutting rate 750 cuts/min and lowaspiration 150 mmHg (Fig. 1b). The aspiration wasreduced when getting closer to the retina and posteriorvitreous detachment was attempted (Fig. 1c). Retinalperiphery was then indented and the vitreous baseremoved (Fig. 1d). In groups with immediate vitrectomy,the infusion cannula was applied before the induction oftrauma to maintain adequate intraocular pressure (IOP) foreasy scleral incision and suturing. The infusion was closed

at the time of blood injection. The procedure was stoppedfor 20 min to allow the blood to coagulate. At the end ofthe surgery, the retina was examined for the presence ofretinal breaks or detachment. The number of the iatrogenicretinal breaks was recorded. If the retina was attached, notamponade was used. In the presence of retinal breaks,only fluid–air exchange was done. The sclerotomies wereclosed followed by closure of the conjunctiva and thelateral canthotomy. At the end of the surgery, an antibioticeye ointment (Refobacin®; Merck KgaA, Darmstadt,Germany) was applied once daily and continued for a

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week. The anterior segment and the fundus were examinedat weekly intervals (Fig. 2a).

Preparation and administration of decorin

Decorin (D-8428; Sigma-Aldrich, Steinheim, Germany)was dissolved in phosphate buffered saline (PBS)(0.5 mg/0.25 ml). The drug was diluted to reach a finaldose of 100 μg/0.1 ml, in GIII and 200 μg/0.1 ml, in GIV.A 30-gauge needle was used to inject 0.1 ml intravitreallyafter the vitrectomy was completed.

Histopathological examination

On the 30th post-vitrectomy day, while the animal wasunder general anesthesia, the right eye was enucleated. Adose of 0.3 ml/kg T-61 (a combination of embutramide,mebenzoniumiodide, and tetracaine hydrochloride; HoechstRoussel Vet, Frankfurt, Germany) was then injected throughthe intracardiac route. The whole eye was fixed for 36–48h in a mixture of 1% buffered formaldehyde and 1.25%glutaraldehyde. The eyes were then processed for staining

with hematoxylin and eosin and Masson’s trichrome stainsas previously described [34].

Photographs

A consistent clinical observation of the fundus was notpossible in all animals due to media opacities as cataract,vitreous hemorrhage, the development of fibrous ingrowth,the development of postoperative iritis, and poorly dilatedpupils. A detailed and reliable anatomic evaluation wastherefore preformed on the enucleated eye under an operatingmicroscope (Zeiss OPMI, Jena, Germany). Clinical and grosspathology findings were recorded with a camera (Sony CCD,DXC-107, Tokyo, Japan) attached to the operating micro-scope. Photographs were then captured from the video recordswith ACDSee Pro software version 8.1 (ACD system, Ltd,British Columbia, Canada). The external appearance of thesite of the trauma was first documented. The eyes weresectioned through the midpoint of the wound. The resultedcalottes were examined and photographed. Histological speci-mens were examined and documented with inverted micro-scope (Leica DMI 6000 B microscope, Wetzlar, Germany).Photos were captured using a DFC 290 compatiblecamera and the appropriate software (Leica ApplicationSuit LAS Software, Wetzlar, Germany).

Image analysis for fibrosis evaluation

The severity of PVR was staged into five stages based onthe grading system described by Cardillo et al. [35](Fig. 2b–f). Photographs of the external appearance of thepost-traumatic wound were evaluated and the maximumwidth of the healed wound was calculated with the 1Dmeasurement tool of the software (JMicroVision©, Univer-sity of Geneva) as previously described [36] (Fig. 2g). As apreparatory step for morphometric analysis of fibrosis,Masson's trichrome stain was used to distinguish cells fromsurrounding connective tissue. Collagen fibers of theconnective tissue were stained blue. A careful morphomet-ric analysis of developed fibrosis at the site of the woundwas carried on three randomly selected 640× photographs.Photographs were analyzed with the Magic Wand tool ofthe same software. Areas stained in blue were selectedbased on pixels similarity then measurements for the area(μm2) were calculated [36] (Fig. 2h and i).

Statistical evaluation

Statistical analysis was performed using SPSS 16 software(SPSS Inc, Chicago, IL, USA). Descriptive analysis for theresults was reported as the median and the interquartilerange for non-parametric parameters, and as mean andstandard deviation for parametric one. The Mann–Whitney

Fig. 2 PVR score and fibrosis assessment. a Clinical evaluation (GI,case number 2): Fundus examination revealed PVR development PVRstage 3 (large arrow) with traction over the medullary ray (smallarrow). b PVR stage 4 (GI, case number 4): 100% of the retina wasdetached associated with a closed funnel configuration. Fibrousingrowth (arrow) was severe, with obvious dragging of the retinacausing prominent retinal folds with viscous subretinal fluid formation(star). The lens was cataractous and pushed anteriorly by the fibrousmembrane. c PVR stage 3 (GIIc, case number 3): Between 50 and100% of the retina (large arrow) was detached (severe retinaldetachment), associated with an open-funnel configuration (smallarrows). Fibrous ingrowth from the wound was severe. A viscoussubretinal fluid (star) was formed. In this case, 100% of the retina wasdetached. d PVR stage 2 (GIIIc, case number 4): Up to 50% of theretina was detached (moderate tractional elevation), usually directlysurrounding the ray (arrows). Prominent fibrous ingrowth was present,with faint bands connecting the peripheral ray fibrous mass to the discor ray. In this case, detachment was minimal and confined to themedullary ray. e PVR stage 1 (GIIIb, case number 2): The retina isattached with minimal traction elevation confined to the medullary ray(arrows). f PVR stage 0 (GIVb, case number 2): Attached retina(arrows) without evidence of fibrous ingrowth. g Estimation of theexternal wound breadth (GI, case number 2): The maximum breadth(mark) of the healed wound (arrow) was calculated with the 1Dmeasurement tool of the software (JMicroVision©, University ofGeneva). h Estimation of the developed fibrosis at wound site (a)(GIIc, case number 3): As a preparatory step for morphometricanalysis of fibrosis, Masson's trichrome stain was used to distinguish-ing cells from surrounding connective tissue. Collagen fibers of theconnective tissue were stained blue (arrow). i Estimation of thedeveloped fibrosis at wound site (b) (GIIc, case number 3): Amorphometric analysis of developed fibrosis at the site of the woundwas carried on 640× photographs. Photographs were analyzed withthe Magic Wand tool of the software (JMicroVision©, University ofGeneva). The color tolerance was adjusted to include a wide range ofthe blue color intensity and then these different degrees were mergedand the total area (arrow) was calculated (μm2)

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Fig. 3 Effects of the adjuvant intravitreal use of decorin followingvitrectomy. a PVR stage 0 (GIVa, case number 4): The retina (whitearrows) was completely attached. Minimal fibrous strands were seenover the medullary ray (black arrow). b PVR stage 0 (GIVb, casenumber 5): The retina (white arrows) was completely attached withoutevidence of fibrous traction. The medullary ray showed no fibroustraction (black arrow). c Attenuated fibrous ingrowth (GIIIa, casenumber 2): Fibrous ingrowth entangling blood (large white arrow)was seen arising from the site of trauma (small white arrow). Theingrowth was markedly attenuated and extends to the remains of thelens (star). d Attenuated PVR (GIIIc, case number 1): Fine fibrousstrands (white arrows) were seen stretched between small retinal folds(black arrows). e PVR formation in the presence of retinal tears,(GIIIb, case number 4): The retina (white arrows) was partially

detached despite the presence of a retinal break (black arrow), aviscous subretinal exudates (star) was formed. f PVR formation in thepresence of retinal tears, (GIVc, case number 1): The retina (whitearrows) was completely attached in spite of the presence of retinalbreak (black arrow). No PVR reaction was detected. g Estimation ofthe external wound breadth (GIV, case number 2): The maximumbreadth (mark) of the healed wound (arrow) was calculated with the1D measurement tool of the software (JMicroVision©, University ofGeneva). h Estimation of the developed fibrosis at wound site, (GIIIc,case number 3): Collagen fibers of the connective tissue were stainedblue (black arrow). i Estimation of the developed fibrosis at woundsite (b), (GIIIc, case number 3): Measurement of the fibrous area(arrow) using the Magic Wand tool of the software (JMicroVision©,University of Geneva)

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non-parametric test was used to compare median values ofdifferent variables between groups. For all tests, the level ofsignificance was set at p

kidney [45], lung [46], cerebral [47], and muscular fibrosis[48]. In ocular diseases, decorin was evaluated as anti-fibrotic agent following glaucoma filtering surgery [28]. Arecent study showed that decorin gene transfer effectivelyprevents TGF-ß driven transformation of keratocyte andcorneal fibroblast to myofibroblasts [49].

Because the mechanisms involved in the pathogenesis ofPVR may differ in rhegmatogenous RD compared totrauma, specific PVR animal models of ocular penetratingtrauma have been developed in non-human primates [30,50, 51] and rabbits [29, 32, 52, 53]. These models allow theevaluation of complicated ocular injury in a controlledmanner to elucidate mechanisms of injury, cellular prolif-eration and tractional forces leading to TRD and PVR. Wehave used a previously described rabbit model of traumaticPVR [29, 52]. In this study, two modifications wereintroduced to the used model to improve its reliability.First, with standardized oblique scleral incision, the modelwas able to produce constant severe forms of PVR andTRD, which simulate the human condition (Fig. 2b).Second, we used a fixation mixture of (1% bufferedformaldehyde/1.25% glutaraldehyde for 36–48 h) for fixing

whole eyes. This solution resulted in minimal volumecontraction without compromising cellular preservation[54]. Consequently, the reliability of the results wasimproved. In turn, it avoided the inconsistency of observa-tions based on clinical fundus examination. As demon-strated in control group GI, the model was effective andreliable. Clinical landmarks describing the progression ofexperimental PVR have been divided into those thatfeature a model using an intact vitreous [55–57] andthose that include compressed vitreous as a step in thedevelopment of PVR [58]. However, both classificationsdescribed the developed PVR in vivo non-traumaticrabbits PVR models and do not fit to describe our results.To overcome this problem, a PVR scoring system based onthe natural history of penetrating ocular trauma in rabbitwas used [32, 35, 52]. A PVR stage of 1 or less was usedto indicate effective treatment.

The multiple features of acute ocular injury make it verydifficult to interpret retrospective data regarding the mostappropriate timing for surgical intervention [59–62].Performing vitrectomy under fixed experimental conditionsallows better understanding of the outcome. Cleary and

Table 4 Comparison between decorin-treated groups and the control group

GI and GIIIa GI and GIIIb GI and GIIIc GI and GIII GI and GIVa GI and GIVb GI and GIVc GI and GIV

PVR score 0.033* 0.013* 0.049* 0.005* 0.009* 0.003* 0.05* 0.001*

Wound size 0.009* 0.175 0.117 0.021* 0.009* 0.009* 0.009* 0.001*

Fibrosis area 0.009* 0.016* 0.016* 0.002* 0.016* 0.016* 0.047* 0.005*

*Significant p value, (Mann–Whitney test)

Table 3 Descriptive analysis of the different evaluation parameters

Retinal breaks PVR score Wound width (μm) Fibrosis area (μm2)

M P M P Mean SD Mean SD

M 25 75 M 25 75

GI 0 0 0 3 1.5 3.5 68 17 5,734 603

GIIa 0 0 2 0 0 2 78 13 9,639 2,470

GIIb 0 0 1 3 2 3 68 12 6,180 1,062

GIIc 0 0 1.5 3 2 4 58 18 6,924 825

GII 0 0 2 3 0 3 68 16 7,581 2,151

GIIIa 2 0 2.5 1 0 2 101 9 2,313 319

GIIIb 0 0 1 0 0 1 81 9 3,355 1,250

GIIIc 0 0 1 0 0 2 91 16 4,389 565

GIII 0 0 2 0 0 1 91 14 3,352 1,156

GIVa 0 0 0 0 0 0 186 31 2,335 1,910

GIVb 0 0 2 0 0 1 176 14 4,279 594

GIVc 1 0 1.5 1 0 2 209 17 4,572 437

GIV 0 0 2 0 0 1 190 25 3,729 1,501

PVR proliferative vitreoretinopathy, M median value, P percentiles, SD standard deviation

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Ryan used such a model to evaluate vitrectomy inexperimental posterior penetrating eye injury in the rhesusmonkey [51]. Cryopexy, scleral buckling, and sulfurhexafluoride tamponade were used when indicated. Theyconcluded that vitrectomy at 1 or 14 days after traumasignificantly prevents TRD. In the current study, we choseto combine this traumatic PVR model and 23-gauge pars

plan vitrectomy. The use of 23-gauge vitrectomy greatlyshortens the procedure time. However, the trocars should beobliquely inserted to prevent the instability of the vitrectomycannulas caused by thin rabbit’s sclera; as well as to improvethe sealing of the wounds. In our study, no retinopexy,buckling, or internal tamponade except for air were used. Wetried to limit the number of the factors that might influence the

Fig. 4 Histopathology. a Fi-brous reaction following trauma(GI, case number 1): The lenshas been lost during processing.Fibrous proliferation (large arrow)from the wound (star) appears tobe derived mainly from the uvealtract. The retina (small arrow) isdrawn up to the wound by thisfibrous proliferation. The fibroustissue was in the process offorming a cyclitic membrane(Masson’s trichrome stain, 50×). bFibrous reaction following trauma(GI, case number 3): Another eyeshowing severe PVR develop-ment after injury. The wound(star) is organized and has bloodvessels and scattered pigment inthe fibrous tissue (large blackarrow). A marked fibrous prolif-eration extends inwards from thewound and swirls of fibrous tissueform a plaque on the surface ofthe retina (long arrow), (Masson’strichrome stain, 50×). c Fibrousreaction following decorin treat-ment (GIIIb, case number 2): Aweak fibrous proliferation (largearrow) extends inwards from thewound (star). The adjacent retina(small arrow) was seen attached(Masson’s trichrome stain, 50×). dAborted PVR formation (GIIIa,case number 4): An epiretinalmembrane was present with min-imal collagen deposition (arrow)while the retina was attached(star) (Masson’s trichrome stain,100×). e Attached retina, (GIIIb,case number 5): Low magnifica-tion image of the retina (arrow)showed perfectly attached retinawith preserved architecture (Mas-son’s trichrome stain, 50×). fNormal photoreceptor layer: Ahigh magnification image of theretina showed preserved details ofthe outer nuclear layer (whitearrow) and the retinal photore-ceptors layer (black arrow). Thephotoreceptors–retinal pigmentepithelium relationship was pre-served (Masson’s trichrome stain,1,600×)

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outcome, thus a real evaluation of the given treatment couldbe achieved. We found that vitrectomy done immediatelyfollowing trauma at the end of the follow-up time significantlyreduced the PVR score compared to the control (p

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The novel use of decorin in prevention of the development of proliferative vitreoretinopathy (PVR)AbstractAbstractAbstractAbstractAbstractIntroductionMaterials and methodsAnimalsAnesthesiaSurgical procedurePreparation and administration of decorinHistopathological examinationPhotographsImage analysis for fibrosis evaluationStatistical evaluation

ResultsEffects of trauma (GI)Effects of vitrectomy as single treatment (GII)Effects of adjuvant decorin therapy (GIII and GIV)Histopathology

DiscussionReferences