internal limiting membrane ( ilm ) , the basement membrane of retinal mller cells , is the structural interface between the vitreous and retina .
histologically , ilm tissues isolated from patients with macular holes ( mhs ) revealed amorphous linear structure in which glial cells , hyalocytes , and myofibroblast - like cells could be intermingled.1,2 today , brilliant blue g ( bbg ) is well known to be a useful agent to stain ilm with blue coloration and to facilitate ilm peeling.3 ilm removal in mh surgeries relieves tangential traction around the holes , increases mh closure rates , and contributes to reduced frequency of epiretinal membrane formation.4 thus , ilm peeling during vitrectomy has become one of the pivotal procedures in surgical intervention for mhs .
however , vitreoretinal surgeons can not always close every mh using vitrectomy with conventional ilm peeling .
especially , surgical interventions for large mhs , measuring > 400 m in the base diameter , and for high - myopic mhs are challenging because of their low closure rates.5 sometimes , such holes could be closed with exposure of the retinal pigment epithelium to the vitreous space , resulting in postoperative poor visual acuity.6 in order to improve the hole closure rate , inverted ilm flap technique has recently been applied to patients with refractory mhs .
indeed , this technique includes isolation of ilm tissues , which are not completely peeled off but placed on the hole .
this technique is also useful for closure of a large mh secondary to uveitis.7 however , it is largely unknown whether it is possible to use bbg - negative membranes as materials for autologous transplantation in order to facilitate the hole closure .
in addition , it has not been clarified how the inverted ilm flap technique leads to morphological changes following the mh closure and to postoperative visual outcomes .
the aim of this study is to analyze optical coherence tomography ( oct ) imaging of large mhs treated with inverted ilm flap technique .
this study further performed a histological examination of an ilm - like membrane tissue obtained during vitrectomy .
the diagnostic criterion for large mh includes the minimum diameter of the holes > 400 m determined by oct imaging at an initial presentation .
high - myopic mh is identified as follows : spherical equivalent > 7.0 d and the ocular axis > 26 mm determined by a - scan ultrasonography and/or intraocular lens ( iol ) master .
this study also excluded cases showing a minimum mh diameter of < 400 m , the existence of proliferative vitreoretinopathy or other retinal breaks , mh retinal detachment , and medical history of having ocular surgeries .
all patients underwent 23 g or 25 g pars plana vitrectomy ( ppv ) with / without phacoemulsification and iol implantation , conducted by sk and ws in hokkaido university hospital or teine keijinkai hospital between june 2013 and march 2015 .
if the posterior vitreous detachment ( pvd ) was not present , the surgeon manually conducted artificial pvd formation .
after visualization of ilm by bbg staining , ilm was peeled but the ilm around mh was not completely removed .
the ilm tissues were left as much as possible , which were placed within the hole based on the previous reports.8,9 the ilm tissues beyond the hole within the arcade were then peeled off .
sf6 gas , with the concentration of 16%20% , was injected following fluid / air exchange .
postoperative ophthalmological findings , including best - corrected visual acuities , dilated fundus examination , and oct imaging ( cirrus hd - oct model 4000 [ carl zeiss meditec , osaka , japan ] ) , were recorded before and 1 , 3 , and 6 months after surgery .
this study was approved by the institutional review board ( irb ) of hokkaido university graduate school of medicine ( irb number : 15 - 0068 ) , and the study adhered to tenets set forth in the declaration of helsinki .
the ilm tissue was excised during vitrectomy , which was immediately fixed with 4% paraformaldehyde in an operating room .
formalin - fixed , paraffin - embedded tissue sections of 5 m thickness were made .
the slides were dewaxed , rehydrated , and rinsed in phosphate - buffered saline ( pbs ) twice for 10 minutes and were submitted for hematoxylin and eosin staining and periodic acid schiff ( pas ) staining to confirm the presence of ilm .
, microwave - based antigen retrieval was performed in 10 mm citrate buffer ( ph 6.0 ) .
these slides were incubated with 3% hydrogen peroxide for 10 minutes and then with normal goat serum for 30 minutes .
sections were incubated with anti - glial fibrillary acidic protein ( gfap ; dilution 1:50 ; dako , tokyo , japan ) antibody at 4 overnight .
slides were examined using a keyence bz-9000 ( keyence , osaka , japan ) microscope .
wilcoxon - signed rank test was applied for the evaluation of differences in visual acuities before and after the surgeries at the final presentation .
the diagnostic criterion for large mh includes the minimum diameter of the holes > 400 m determined by oct imaging at an initial presentation .
high - myopic mh is identified as follows : spherical equivalent > 7.0 d and the ocular axis > 26 mm determined by a - scan ultrasonography and/or intraocular lens ( iol ) master .
this study also excluded cases showing a minimum mh diameter of < 400 m , the existence of proliferative vitreoretinopathy or other retinal breaks , mh retinal detachment , and medical history of having ocular surgeries .
all patients underwent 23 g or 25 g pars plana vitrectomy ( ppv ) with / without phacoemulsification and iol implantation , conducted by sk and ws in hokkaido university hospital or teine keijinkai hospital between june 2013 and march 2015 .
if the posterior vitreous detachment ( pvd ) was not present , the surgeon manually conducted artificial pvd formation .
after visualization of ilm by bbg staining , ilm was peeled but the ilm around mh was not completely removed .
the ilm tissues were left as much as possible , which were placed within the hole based on the previous reports.8,9 the ilm tissues beyond the hole within the arcade were then peeled off .
sf6 gas , with the concentration of 16%20% , was injected following fluid / air exchange .
postoperative ophthalmological findings , including best - corrected visual acuities , dilated fundus examination , and oct imaging ( cirrus hd - oct model 4000 [ carl zeiss meditec , osaka , japan ] ) , were recorded before and 1 , 3 , and 6 months after surgery .
this study was approved by the institutional review board ( irb ) of hokkaido university graduate school of medicine ( irb number : 15 - 0068 ) , and the study adhered to tenets set forth in the declaration of helsinki .
the ilm tissue was excised during vitrectomy , which was immediately fixed with 4% paraformaldehyde in an operating room .
formalin - fixed , paraffin - embedded tissue sections of 5 m thickness were made .
the slides were dewaxed , rehydrated , and rinsed in phosphate - buffered saline ( pbs ) twice for 10 minutes and were submitted for hematoxylin and eosin staining and periodic acid schiff ( pas ) staining to confirm the presence of ilm .
the slides were further submitted for immunohistochemistry . as a pretreatment , microwave - based antigen retrieval was performed in 10 mm citrate buffer ( ph 6.0 ) .
these slides were incubated with 3% hydrogen peroxide for 10 minutes and then with normal goat serum for 30 minutes .
sections were incubated with anti - glial fibrillary acidic protein ( gfap ; dilution 1:50 ; dako , tokyo , japan ) antibody at 4 overnight .
slides were examined using a keyence bz-9000 ( keyence , osaka , japan ) microscope .
wilcoxon - signed rank test was applied for the evaluation of differences in visual acuities before and after the surgeries at the final presentation .
the patients age ranged from 51 to 71 years ( mean : 64.8 years ) .
all the nine patients revealed large mhs , consisting of five idiopathic mhs , one large mh secondary to branch retinal vein occlusion , and three high - myopic mhs .
eight patients underwent ppv together with phacoemulsification and iol implantation , while one patient already had a pseudophakic eye before surgery .
ilm was clearly stained with bbg in eight patients ( figure 1a  d ) , whereas the ilm in case 2 ( table 1 ) revealed no staining with bbg even after completion of artificial pvd formation ( figure 1e  g ) .
the follow - up periods ranged from 4 to 28 months ( mean : 16.1 months ) .
preoperative and postoperative best - corrected visual acuities at the final visit ( logmar ) were 0.900.38 and 0.570.41 , respectively .
there was no significant improvement in postoperative visual acuity ( p=0.10 ) , although visual acuities improved to > 0.2 logmar in six patients .
oct findings were available 13 days after the surgery under gas tamponade in three eyes , in which all the patients examined showed findings suggestive of favorable closure .
the complete closure of mh following ppv with inverted ilm technique was eventually achieved in all patients determined by oct imaging ( 100% ) by 1 month after surgery . in this study , after ppv with inverted ilm flap technique , the closure type was u - type ( figures 1d , h and 2a , b ) and v - type ( figure 2c and d ) in eight eyes and one eye , respectively .
oct detected linear configuration , consistent with transplanted ilm tissues , in four eyes 1 month after the surgery .
three out of nine eyes showed elongation of outer nuclear layer 3 months after the surgery .
only one patient ( case 6 in table 1 ) showed recovery of ellipsoid zone and interdigitation zone , and elongation of photoreceptor outer segment length following the surgery .
there are no significant changes in oct finding between 3 and 6 months after the surgery .
the tissue of an ilm - like membrane was isolated during ppv in one patient with stage iii mh ( case 2 in table 1 ) .
the ilm - like membrane not stained with bbg revealed an eosinophilic amorphous structure in conjunction with mononuclear cell infiltration ( figure 3a ) .
pas staining showed negative for cell infiltration but positive for the amorphous structure , which is consistent with ilm ( figure 3b ) .
oct findings were available 13 days after the surgery under gas tamponade in three eyes , in which all the patients examined showed findings suggestive of favorable closure .
the complete closure of mh following ppv with inverted ilm technique was eventually achieved in all patients determined by oct imaging ( 100% ) by 1 month after surgery . in this study , after ppv with inverted ilm flap technique , the closure type was u - type ( figures 1d , h and 2a , b ) and v - type ( figure 2c and d ) in eight eyes and one eye , respectively .
oct detected linear configuration , consistent with transplanted ilm tissues , in four eyes 1 month after the surgery .
three out of nine eyes showed elongation of outer nuclear layer 3 months after the surgery .
only one patient ( case 6 in table 1 ) showed recovery of ellipsoid zone and interdigitation zone , and elongation of photoreceptor outer segment length following the surgery .
there are no significant changes in oct finding between 3 and 6 months after the surgery .
the tissue of an ilm - like membrane was isolated during ppv in one patient with stage iii mh ( case 2 in table 1 ) .
the ilm - like membrane not stained with bbg revealed an eosinophilic amorphous structure in conjunction with mononuclear cell infiltration ( figure 3a ) .
pas staining showed negative for cell infiltration but positive for the amorphous structure , which is consistent with ilm ( figure 3b ) .
in this study , ppv with inverted ilm flap technique achieved 100% closure rates at the initial surgery in nine patients with large mhs .
michalewska et al8 first described the initial closure rate of 98% by inverted ilm flap technique in the patients with large mh .
kuriyama et al5 reported primary closure following the surgery in five eyes out of six eyes with highly myopic mh .
imai et al6 proposed oct - based evaluation of mh closure following vitrectomy . briefly , u - type closure is the most favorable closure type possibly leading to good postoperative visual acuity .
next , v - type shows notch configuration covered with thin retinal tissue . finally , w - type is not favorable closure , so - called open - closed mh , exposing retinal pigment epithelium to the vitreous space .
this case series also proved ideal u - type closure , a successful closure configuration , in most eyes using the inverted ilm flap technique . in this study , the ilm - like membrane
was partially removed in one eye showing no staining with bbg , which was submitted for pathological analyses .
histologically , the ilm - like membrane contained an amorphous linear structure that is pas positive , which is typically observed in the ilm tissue .
uemoto et al10 reported that bbg - positive ilm isolated from mh and epiretinal membrane revealed a membrane with few cell infiltration based on pathological analysis .
therefore , the bbg - negative case shown in this report may represent more glial cell infiltration within the ilm tissue than ilm stained with bbg . as shown in figure 1 , the postoperative oct findings were similar to those transplanted by common bbg - positive ilm tissues .
kuriyama et al5 reported that epiretinal membranes / ilm tissues were transplanted in the hole during vitrectomy in three eyes among six high myopic eyes .
taken together , our data also suggest that even bbg staining - negative ilm - like membrane can be safely used as a material for autologous transplantation to the hole .
it has been speculated that ilm peeling may induce retinal gliosis , leading to the formation of a bridge between the deficit of retinal tissues in the hole.8 this study indicated that the ilm - like membrane including gfap - positive retinal glial cells contributed to the hole closure with a favorable configuration determined by oct .
these results suggest that glial cells placed on the hole may produce intermediate filaments and provoke tissue remodeling within the mh .
the oct demonstrated no recovery of deficit of ellipsoid zone and no elongation of photoreceptor outer segment length in eight out of nine patients examined , although six patients showed improvement of > 0.2 logmar visual acuities .
de sisternes et al11 addressed that recovery of visual acuity was correlated with preoperative degree of the ellipsoid zone deficit in patients with mh .
we recently showed that elongation of photoreceptor outer segment length following mh surgery correlated with postoperative visual acuities.12 however , these results are not consistent with the present study , indicating that the recovery of visual acuity did not depend on preoperative severity of ellipsoid zone and photoreceptor damage in patients with large mh treated with inverted ilm flap technique . in this study
, elongation of outer nuclear layer and external limiting membrane was noted in a few patients , indicating that alteration of visual fixation may correlate with the recovery of visual acuity following the surgery in selected cases .
further studies are needed to analyze the mechanisms underlying postoperative recovery of visual acuity in inverted ilm flap technique .
ppv with inverted ilm flap technique achieved 100% closure rates with favorable configuration at an initial surgery in large mhs .
our data also suggest that even bbg staining - negative membrane may be one of useful materials for autologous transplantation to the hole .
 purposethe aims of this study were to analyze optical coherence tomography ( oct ) imaging of large macular holes ( mhs ) treated with inverted internal limiting membrane ( ilm ) flap technique and to perform a histological examination of an ilm - like membrane tissue obtained during vitrectomy.patients and methodsthis is a retrospective observational case study . 
 nine patients , comprising of five males and four females , showing large and myopic mhs , underwent pars plana vitrectomy ( ppv ) with inverted ilm flap technique assisted by brilliant blue g ( bbg ) staining . 
 ophthalmological findings including visual acuity and oct were investigated based on medical records . 
 formalin - fixed paraffin - embedded tissue section of an ilm - like membrane was submitted for immunohistochemistry with glial fibrillary acidic protein ( gfap).resultsilm was clearly stained with bbg in eight patients , whereas the ilm in one case revealed no staining with bbg during ppv . 
 visual acuities improved to > 0.2 logmar in six patients . 
 the complete closure of mh following ppv with inverted ilm technique was eventually achieved in all patients determined by oct imaging ( 100% ) . 
 only one patient showed recovery of ellipsoid zone and interdigitation zone following the surgery . 
 elongation of outer nuclear layer was noted in three eyes . 
 the ilm - like membrane not stained with bbg histologically revealed an amorphous structure admixed with gfap - positive mononuclear cell infiltration.conclusionppv with inverted ilm flap technique achieved 100% closure rates with favorable configuration at an initial surgery in large mhs . 
 our histopathological data also suggest that even bbg staining - negative membrane may be a useful material for autologous transplantation to the hole . 
Introduction
Patients and methods
Patients
Histology and immunohistochemistry
Statistical analysis
Results
Changes in OCT findings before and after surgery
Histological findings
Discussion
Conclusion