recently , the term of lamellar hole - associated epiretinal proliferation ( lhep ) was introduced by pang and colleagues to characterize a thick homogenous layer of unusual material on the epiretinal surface in eyes with lamellar macular holes ( lmh ) [ 1 , 2 ] . high - resolution optical coherence tomography ( oct ) studies have demonstrated that eyes with lmh frequently show this epiretinal proliferation presenting as a highly reflective line with moderately reflective material filling the space between the inner border of the epiretinal proliferation and the retinal nerve fibre layer [ 37 ] . however , pathogenesis , morphology , and clinical course of eyes with lhep are poorly understood . by recent oct studies , the presence of lhep was shown to be related to the presence of photoreceptor layer defects and poor visual acuity compared to eyes with lmh without lhep [ 1 , 2 , 8 ] . on oct examination , lhep does not appear to have contractive properties [ 1 , 2 , 5 , 8 ] . in general , traction forces by conventional erm become visible as retinal folds that are usually not seen in retinal layers covered by lhep . since there is little detail on cell and collagen composition of lhep , the aim of this study was to describe morphologic characteristics of lhep in eyes with lmh by correlative microscopy . correlative light and electron microscopy ( clem ) was recently proposed using immunonanogold particles of 1.4  nm diameter combined with a fluorescein moiety that both are attached to a single antibody fragment [ 911 ] . in this study surgically excised specimens of lhep were consecutively harvested from 10 eyes of 10 patients with lamellar macular holes during vitrectomy . all patients required surgery due to the severity of clinical symptoms such as progressive visual loss . patients ' records were reviewed for age , gender , previous ocular surgery , and preoperative history such as trauma . all specimens were obtained from patients who were part of a recent retrospective oct study reporting on clinical course of 112 operated and nonoperated eyes with lamellar macular holes and macular pseudoholes . clinical data of patients and volume b - scans of high - resolution oct examinations ( spectralis oct , heidelberg engineering , heidelberg , germany ) were reevaluated in order to exclusively include eyes with presence of lhep . this study was approved by the institutional review board and ethics committee and was conducted according to the tenets of the declaration of helsinki . surgery was recommended if bcva decreased to logmar 0.3 or more , if bcva decreased 2 snellen lines or more during the preoperative follow - up period , and if the patient experienced a significant impairment of the quality of life . if necessary , a posterior vitreous detachment ( pvd ) was induced by suction with the vitrectomy probe around the optic nerve head . the posterior hyaloid was detached from the retina and pvd was extended to the periphery with the vitreous being removed at least up to the equator . for removal of lhep , intraocular end - gripping forceps were used . for ilm peeling , a 0.25  mg / ml solution of brilliant blue ( brilliant peel , fluoron gmbh , neu - ulm , germany ) was used . the vitreous cavity was then filled with a tamponade of either 15% hexafluoroethane ( c2f6 ) gas - air mixture , or air , or balanced salt solution . infrequently , patients were instructed to keep a face - down position for at least 2 days . for fixation indirect immunocytochemistry was performed for all specimens after flat - mount preparation following interference and phase contrast microscopy . if necessary , large specimens were divided into pieces in accordance with their size in order to label excised tissue of each patient with all antibodies . primary antibodies for glial and retinal cells ( antiglial fibrillary acidic protein ( anti - gfap ) , dako , hamburg , germany ) ; for hyalocytes ( anti - cd45 and anti - cd64 , santa cruz biotechnology , heidelberg , germany ) ; for myofibroblasts ( anti--smooth muscle actin ( anti--sma ) , santa cruz biotechnology , heidelberg , germany ) ; and for extracellular matrix ( anticollagen type i ( anti - col - i ) , santa cruz biotechnology , heidelberg , germany ; anticollagen type ii ( anti - col - ii ) , biotrend , cologne , germany ) were added and incubated over night at room temperature . as second antibody , fluoronanogold ( fab-fragments , nanoprobes , yaphank postfixation with 2% glutaraldehyde was following . preparing negative controls , specimens with large area and multilayered cell proliferation were cut into half in order to use one part for labelling procedures and the other part for negative control preparation . the primary antibody was substituted with both diluent and isotype controls ( igg2a monoclonal mouse antibodies , x0934 , dako , hamburg , germany ; m5409 , sigma - aldrich , taufkirchen , germany ) . following fluorescence microscopic analysis ( leica dm 2500 , wetzlar , germany ) at magnifications between 50 and 400 , specimens were processed for transmission electron microscopy . postfixation in osmium tetroxide 2% and uranyl acetate as well as dehydration and embedding in epon 812 was following . ultrathin sections of 60  nm were obtained by series - sectioning and were contrasted with uranyl acetate and lead citrate . analysis and imaging of 5 grids ( each with 69 ultrathin sections ) per specimen were performed using a transmission electron microscope zeiss em 9 s-2 ( zeiss , jena , germany ) . this is a series of 10 surgically excised specimens of lhep obtained during vitrectomy from eyes with lamellar macular holes . patients ' mean age was 70  6 years ( median , 70 years ; range , 6382 years ) . preoperatively median bcva of eyes with lmh was logmar 0.40 ( mean 0.41  0.13 sd ) and increased postoperatively to median bcva of logmar 0.30 ( mean 0.34  0.19 sd ) during a mean follow - up period of 8.6 months ( median 10 months ; range , 315 months ) . in detail , 6 of 10 patients improved visual acuity , whereas 3 of 10 patients lost vision , and in one patient bcva remained unchanged . from the eyes with lhep only , 4 of 5 eyes improved bcva , whereas one of 5 patients lost vision acuity . considering eyes with a combination of both lhep and an extrafoveal conventional erm , 2 of 5 patients improved vision acuity . two of 5 patients lost bcva and one patient was stable . at time of surgery , all of the phakic eyes underwent combined vitrectomy with cataract extraction and intraocular lens implantation . regarding the presence of posterior vitreous detachment ( pvd ) , a complete pvd was seen in 4 of 10 eyes as intraoperatively assessed by the surgeon . a partial pvd was documented in 4 of 10 eyes , and an attached posterior vitreous was found in 2 of 10 eyes . postoperatively , none of the eyes developed a full - thickness macular hole and no persistent macular edema was noted . in sd - oct examinations , lhep was directly located at the macular defect ( figures 1(a ) and 1(b ) ) . in half of all eyes , a combination of both lhep and a conventional erm with contractive properties was seen . if present , conventional erm was found extrafoveal with some distance to the foveal defect ( figure 1(c ) ) . preoperatively , defects of the ellipsoid zone were detected in 8 of 10 eyes ( table 1 ) . in 2 eyes , defects of the external limiting membrane ( elm ) analysing flat - mounted specimens , positive immunostaining for anti - gfap and for the hyalocyte cell markers anti - cd45 and anti - cd64 was seen in all eyes with lhep ( table 1 , figure 2 ) . moreover , a colocalisation of anti - gfap with anti - cd64 as well as anticollagen type i was seen in several specimens . in negative control specimens , , the ilm was characterized by its undulated retinal side and the smooth vitreal side . in epiretinal cell proliferation , fibroblasts and hyalocytes were the predominant cell types ( figure 3 ) . fibroblasts were characterized by their abundant rough endoplasmatic reticulum , prominent golgi complex , and a fusiform shape of the cell body and nucleus . hyalocytes were distinguished by their lobulated cell nuclei , intracellular vacuoles , vesicles , and mitochondria as well as long cell fibers . myofibroblasts containing cell fibers with contractile forces were rarely found . in the collagen matrix , native vitreous collagen ( nvc ) was predominant and identified as major type of collagen . it is characterized by a regular arrangement of fibrils with a collagen fibril diameter of less than 16  nm . newly formed collagen ( nfc ) with irregular fibril arrangement and fibril diameter of more than 16  nm was seen as well . in nvc , fibrous long spacing collagen ( flsc ) negative controls did show neither specific labelling of cellular structures nor extracellular components by immunonanogold labelling . this is the first correlative light and electron microscopic study presenting histopathologic data of lhep by using application of immunonanogold . correlative light and electron microscopy was recently reported to improve visualization of cells and extracellular matrix in epiretinal membranes by using fluoronanogold as secondary antibody . it composes an immunonanogold particle of 1.4  nm diameter that is combined with a fluorescein moiety and a single antibody fab - fragment [ 912 ] . by application of immunonanogold particles , we were able to analyse the same cellular and extracellular components of lhep by fluorescence and electron microscopy . lamellar hole - associated epiretinal proliferation was recently suggested to be primarily driven by a proliferation of mller cells onto the inner retina originating from the middle layers of the retina [ 1 , 2 ] . in this study , we demonstrated gfap - positive cells in lhep . this finding is in accordance with immunohistological results of parolini and colleagues , who also presented cells with positive immunostaining of anti - gfap . in the majority of studies , immunoreactivity for gfap in epiretinal membranes was usually interpreted as an indicator for the presence of glial cells . however , electron microscopy revealed fibroblasts as predominant cell type in this analysis . of note , fibroblasts and hyalocytes were occasionally described to be gfap - positive [ 1418 ] . several species were found to present gfap - positive hyalocytes , including porcine , pectineal , and bovine hyalocyte cell lines . therefore , we hypothesize that vitreous derived cells rather than cells of glial origin may play a major role in pathogenesis of lmh with lhep . in this series , fibroblasts were often seen densely packed in cell agglomerations , mostly situated on vitreous collagen strands . our observations are in accordance with sd - oct examinations demonstrating a thick homogenous layer of unusual material on the epiretinal surface in eyes with lamellar macular holes that does not show contraction signs [ 1 , 2 ] . predominance of vitreous collagen in specimens of lhep was reported by parolini et al . and has been confirmed by this study . native vitreous collagen fibrils were arranged as thick collagen strands often dispersed with fibrous long spacing collagen that is known to represent a remodelling process of vitreous collagen [ 18 , 19 ] . thus , lhep appears to primarily consist of vitreous derived cells proliferating on vitreous collagen strands that are marked by degradation and remodelling of collagen components . in this study , the majority of eyes with lhep showed defects of the ellipsoid zone in preoperative sd - oct examinations , which is in accordance with previous reports on disruptions of the outer photoreceptor layer in lmh [ 2 , 8 ] . furthermore , recent studies demonstrated that the presence of lhep correlated with defects of the ellipsoid zone and the elm layer [ 8 , 20 , 21 ] . however , it is still unknown why eyes with lmh do not respond to erm / ilm peeling as positively as expected [ 6 , 2224 ] . differences in contractive properties of epiretinal cell proliferation might partly explain these postoperative findings and should be taken into consideration when recommending surgical intervention in eyes with lmh . in this study , half of all eyes with lhep were accompanied by eccentric foci of conventional erm . in these cases of lmh , macular surgery might be indicated and surgical outcome of these eyes should be addressed in further studies .
purpose . to describe morphology of lamellar hole - associated epiretinal proliferation ( lhep ) removed from eyes with lamellar macular holes ( lmh ) . methods . based on optical coherence tomography data , 
 10 specimens of lhep were removed from 10 eyes with lmh during standard vitrectomy . 
 specimens were prepared for correlative light and electron microscopy ( clem ) using an immunonanogold particle of 1.4  nm diameter that was combined with a fluorescein moiety , both having been attached to a single antibody fragment . as primary antibodies , we used antiglial fibrillary acidic protein ( gfap ) , anti - cd45 , anti - cd64 , anti--smooth muscle actin ( -sma ) , and anticollagen type i and type ii . results . in lhep , 
 gfap - positive cells possess ultrastructural characteristics of fibroblasts and hyalocytes . 
 they represent the major cell types and were densely packed in cell agglomerations on vitreous collagen strands . 
 epiretinal cells of lhep rarely demonstrated contractive properties as -sma - positive myofibroblasts were an infrequent finding . 
 conclusion . 
 clem indicates that epiretinal cells in lhep might originate from the vitreous and that remodelling processes of vitreous collagen may play an important role in pathogenesis of eyes with lmh .