Muscle Grafts with Doxorubicin Pretreatment Produce "Empty Tubes" in the Basal Laminae, Promote Contentious Maturation of the Regenerated Axons, and Bridge 20-mm Sciatic Nerve Defects in Rats.

BACKGROUND: We newly developed a muscle graft that employs a doxorubicin pretreatment technique. The aims of this study were to reveal the biological and morphological features of the muscle tissue in the second week (Study I), to reveal the regeneration outcomes of functional and kinematic assessments of longer-term follow-up (16 weeks, Study II), and to make assessments of the muscle graft with doxorubicin pretreatment in the critical-sized nerve defect model (20 mm, Study III). METHODS: A total of 26 adult rats were used in this study. Doxorubicin treatment was accomplished by immersion in a doxorubicin solution for 10 minutes followed by a rinsing procedure. The rats were divided into three groups: the muscle graft with and without doxorubicin pretreatment (M-graft-w-Dox and M-graft-w/o-Dox) groups and the autologous nerve graft (N-graft) group. Assays of apoptosis, immunofluorescent histochemistry including CD68 (macrophage marker), scanning electron microscopy (SEM), morphometrical studies of the regenerated axons, nerve conduction studies, and kinematic studies were performed. RESULTS: The M-graft-w-Dox group contained significantly larger numbers of apoptotic cells and CD68-positive cells. SEM revealed the existence of the basal lamina, so called "empty tubes," in the M-graft-w-Dox group. Study II showed contentious maturation of the regenerated axons, especially in the compound muscle action potentials. Study III showed that even at 20 mm, the M-graft-w-Dox group promoted axonal regeneration and functional regeneration. CONCLUSION: The M-graft-w-Dox group showed superior regeneration results, and this easy and short-term procedure can expand the muscle graft clinical indication for the treatment of peripheral nerve defects.

ANATOMICAL AND VISUAL OUTCOMES OF INVERTED INTERNAL LIMITING MEMBRANE FLAP TECHNIQUE VERSUS INTERNAL LIMITING MEMBRANE PEELING IN MYOPIC MACULAR HOLE WITHOUT RETINAL DETACHMENT: A Preliminary Retrospective Study.

PURPOSE: To compare the results of vitrectomy with internal limiting membrane (ILM) peeling and inverted ILM flap for treating myopic macular hole without retinal detachment. METHODS: Twenty-eight eyes of 28 patients undergoing vitrectomy with either ILM peeling (n = 16) or inverted ILM flap technique (n = 12) were included. Outcomes were myopic macular hole closure by optical coherence tomography and visual acuity at 6 months and at the end of follow-up. RESULTS: Closure of myopic macular hole was achieved in 13 eyes (81.2%) of the ILM peeling group and in 11 eyes (91.7%) of the inverted ILM flap group. The median length of follow-up was 18 months in the peeling group and 10.3 in the inverted group. There were not statistically significant differences between restoration of the external limiting membrane, external limiting membrane and ellipsoid zone, and none of both layers between the two groups. The median best-corrected visual acuity (logarithm of minimal angle of resolution) at the end of follow-up was 0.25 (20/35 Snellen) in the peeling group and 0.4 (20/50) in the inverted group (P = 0.027). CONCLUSION: Both techniques were associated with high closure rates of myopic macular hole but the small sample size and the retrospective design prevents any claims of superiority of one technique over the other.

Dynamic intraoperative optical coherence tomography for inverted internal limiting membrane flap technique in large macular hole surgery.

BACKGROUND/OBJECTIVES: To assess the efficacy of dynamic intraoperative spectral-domain optical coherence tomography (iSD-OCT) imaging for inverted internal limiting membrane (ILM) flap technique (IILMFT) in large macular hole (MH) surgery. SUBJECTS/METHODS: Prospective, non-randomized, observational study was conducted on 8 eyes of 7 patients with large, chronic and recurrent MHs, which were treated by pars plana vitrectomy (PPV) with IILMFT. All patients underwent standard pre- and postoperative examination. The iSD-OCT imaging was performed using microscope integrated systems before, during, and after ILM peeling. The iSD-OCT data were post-processed using graphic software and reviewed for tissue behavior and instruments position. RESULTS: The real-time iSD-OCT-assisted IILMFT allowed for real-time imaging of the entire surgery with visualization of the MH, vitreoretinal instruments, and all steps of inverted ILM flap formation. In spite of shadowing created by the steel instruments, it was possible to follow and control the distance between the instrument tips and retinal layers. Dynamic imaging of the surgical maneuvers including ILM peeling and mechanical apposition of MH edges revealed the iatrogenic impact on the retina (depression and appearance of hyporeflective zones). iSD-OCT imaging could confirm the proper position of the inverted ILM flap at the very end of the surgery after fluid-air exchange. CONCLUSIONS: iSD-OCT imaging is an effective tool for learning and performing a well-controlled and safe inverted ILM flap technique in patients with large MH. Clinical significance of the structural iSD-OCT findings has to be further studied.

Folding the internal limiting membrane flap under perfluorocarbon liquid in large, chronic and myopic macular holes.

PURPOSE: The purpose of the present paper is to describe a surgical technique aimed at creating multiple layers of Internal Limiting Membrane (ILM) using Perfluorocarbon Liquid (PFCL) in order to favour the closure of large, chronic and myopic Macular Holes (MH). METHODS: Thirty patients belonging to 3 subgroups: large (> 500 µm), chronic (> 12 months) and myopic (> - 9 diopters), MHs, underwent surgery and completed 6 months follow-up. The ILM was engaged and peeled 360° around the MH, hinged to the rim and folded over the hole. A PFCL bubble spanning the vascular arcades was then injected and the ILM is grasped repeatedly to fold the distal edge towards the MH centre, creating multiple ILM layers over the MH. RESULTS: MH closed in 26/30 cases (86.6%) with no significant difference among subgroups. Vision improved 2.57 ± 1.56 Snellen lines from LogMAR 1.50 ± 1.19 to 1.19 ± 1.32 (p < 0.01). Ellipsoid Zone (EZ) interruption width reduced from 1129 ± 439 µm to 258 ± 507 µm (p < 0.001) and correlated to pattern of MH closure, post-BCVA and line improvement (p < 0.001 in all cases). DISCUSSION: The use PFCL allows multiple ILM layers and resulted in a high closure rate. Pattern of MH closure differs from those previously described leaving a plug of ILM tissue that interrupts retinal architecture often only in the inner layers. PFCL gravity and hydrophobicity displace aqueous while the intensely polar opposite faces of the ILM attract each other. The folded ILM plugs MH and bridges the gap and may help glial cell proliferation and migration.

One-Year Results after Internal Limiting Membrane Flap Transposition for Surgical Repair of Macular Holes with Respect to Microperimetry.

PURPOSE: Internal limiting membrane (ILM) flap transposition in surgical repair of macular holes represents a new technique offering good anatomical success rates with large macular holes. The aim of the study was to examine microperimetric outcomes 1 year after ILM flap transposition for surgical repair of macular holes. METHODS: Patients with idi-opathic macular holes scheduled for 23-G pars plana vitrectomy with ILM peeling, ILM flap transposition, and SF6 tamponade were examined in a prospective case series. Distance corrected visual acuity (DCVA), optical coherence tomography, and microperimetry were measured before and 1 year after surgery. RESULTS: In all eyes, closure of the macular hole could be achieved. While foveal sensitivity improved in 71%, perifoveal sensitivity improved in 86% of the cases. DCVA improved in 83% and was unchanged in 17% of the cases. CONCLUSIONS: Beside visual acuity, foveal and perifoveal macular sensitivity improved, underlining the functional success of surgery after ILM peeling with ILM flap transposition.

Spare some internal limiting membrane for later: free ILM patch and neurosensory retina graft.

PURPOSE: Editorial to De Giacinto et al case report on free autologous neurosensory retina patch. METHODS: Literature review and experts' opinion RESULTS: In the present issue, De Giacinto et al describe a free autologous neurosensory retina patch to close a chronic macular hole. This new technique was made necessary by an extended internal limiting membrane peeling during the first surgery, that prevented grafting a patch of internal limiting membrane when the hole did not close. We hereby review pros and cons of patching a chronic macular hole with an internal limiting membrane patch, as well as the importance of not over-enlarging a peeling. DISCUSSION: Internal limiting membrane patch can be considered in chronic macular holes. It may not be an option in cases of over-enlargement of a previous peel; free autologous neurosensory retina patch may be a valid alternative in such cases.

Microstructural retinal regeneration after internal limiting membrane flap surgery for repair of large macular holes: a 1-year follow-up study.

PURPOSE: To evaluate long-term change in functional and structural outcomes after successful repair of large macular holes (MH) with internal limiting membrane (ILM) flap techniques. METHODS: Eleven consecutive patients were reviewed over a 1-year time period after the successful repair of large MH with ILM flap techniques. SD-optical coherence tomography (SD-OCT) images were taken to assess the anatomical outcome after surgery, while the best-corrected visual acuity (BCVA) was tested using Snellen charts to evaluate the functional outcome. Each patient was evaluated at 1, 6 and 12 months after surgery, respectively. RESULTS: All cases achieved complete anatomical closure. All patients showed a microstructural regeneration of the retina with a decrease in ellipsoid zone defects over the 1-year follow-up. Functionally, as compared to baseline, all of the patients showed improvements in best-corrected visual acuity of 1-4 lines at the final examination after 12 months post-operatively. CONCLUSIONS: Long-term results show further improvement in the best-corrected visual acuity as well as further microstructural regeneration of the retina and decrease in ellipsoid zone defects over time. The exact mechanism, which promotes closure of the macular hole and reconstruction of the ellipsoid zone after internal Limiting Membrane autograft surgery, still remains unknown.

MECHANISM OF "FLAP CLOSURE" AFTER THE INVERTED INTERNAL LIMITING MEMBRANE FLAP TECHNIQUE.

PURPOSE: Recent studies described that in approximately 14% to 16% of cases of macular holes treated with the inverted internal limiting membrane flap technique, the hole was closed only by a thin layer of inverted internal limiting membrane-"flap closure." The aim of this article was to describe the functional and anatomical results in flap closure macular holes and also the mechanism of flap closure. METHODS: A retrospective observational study of 149 eyes of 139 patients treated with vitrectomy using the inverted internal limiting membrane flap technique was reviewed to aggregate eyes with flap closure. Complete ophthalmic examination was performed preoperatively and at 7 days, 1, 3, 6, and 12 months after surgery. RESULTS: Flap closure (Group 1) was noted in 24 eyes and other closure types in 125 eyes (Group 2). The mean minimal and base diameters of the macular holes in the flap closure group were greater than those in Group 2 (P < 0.001). The mean postoperative best-corrected visual acuity in the flap closure group (20/100) was lower than that in Group 2 (20/50) (P < 0.001). CONCLUSION: Flap closure probably enables closure of large macular holes with a higher probability of remaining open without the use of the inverted internal limiting membrane flap technique. Foveal architecture continuously improved.

INTERNAL LIMITING MEMBRANE PEELING VERSUS INVERTED FLAP TECHNIQUE FOR TREATMENT OF FULL-THICKNESS MACULAR HOLES: A COMPARATIVE STUDY IN A LARGE SERIES OF PATIENTS.

BACKGROUND: The inverted flap (IF) technique has recently been introduced in macular hole (MH) surgery. The IF technique has shown an increase of the success rate in the case of large MHs and in MHs associated with high myopia. This study reports the anatomical and functional results in a large series of patients affected by MH treated using pars plana vitrectomy and gas tamponade combined with internal limiting membrane (ILM) peeling or IF. METHODS: This is a retrospective, consecutive, nonrandomized comparative study of patients affected by idiopathic or myopic MH treated using small-gauge pars plana vitrectomy (25- or 23-gauge) between January 2011 and May 2016. The patients were divided into two groups according to the ILM removal technique (complete removal vs. IF). A subgroup analysis was performed according to the MH diameter (MH < 400 µm and MH ≥ 400 µm), axial length (AL < 26 mm and AL ≥ 26 mm), and the presence of chorioretinal atrophy in the macular area (present or absent). RESULTS: We included 620 eyes of 570 patients affected by an MH, 300 patients underwent pars plana vitrectomy and ILM peeling and 320 patients underwent pars plana vitrectomy and IF. Overall, 84.94% of the patients had complete anatomical success characterized by MH closure after the operation. In particular, among the patients who underwent only ILM peeling the closure rate was 78.75%; among the patients who underwent the IF technique, it was 91.93% (P = 0.001); and among the patients affected by full-thickness MH ≥400 µm, success was achieved in 95.6% of the cases in the IF group and in 78.6% in the ILM peeling group (P = 0.001); among the patients with an axial length ≥26 mm, success was achieved in 88.4% of the cases in the IF group and in 38.9% in the ILM peeling group (P = 0.001). Average preoperative best-corrected visual acuity was 0.77 (SD = 0.32) logarithm of the minimum angle of resolution (20/118 Snellen) in the peeling group and 0.74 (SD = 0.33) logarithm of the minimum angle of resolution (20/110 Snellen) in the IF group (P = 0.31). Mean postoperative best-corrected visual acuity was 0.52 (SD = 0.42) logarithm of the minimum angle of resolution (20/66 Snellen) in the peeling group and 0.43 (SD = 0.31) logarithm of the minimum angle of resolution (20/53 Snellen) in the IF group (P = 0.003). CONCLUSION: Vitrectomy associated with the inverted ILM flap technique seems to be effective surgery for idiopathic and myopic large MHs, improving both functional and anatomical outcomes.

INVERTED INTERNAL LIMITING MEMBRANE FLAP TECHNIQUE FOR MACULAR HOLE SURGERY WITHOUT EXTRA MANIPULATION OF THE FLAP.

PURPOSE: To determine whether surgical manipulation steps of the internal limiting membrane (ILM) flap, such as ILM trimmed, ILM tuck inside the hole, ILM massage, are mandatory to obtain satisfactory outcomes for the repair of large stage IV idiopathic macular hole using the inverted ILM flap technique. METHODS: In this interventional comparative prospective single-masked study, 81 eyes were randomized into 2 treatments groups. In Group 1 (41 eyes), the classic inverted ILM flap technique was performed. In Group 2 (40 eyes), a modified procedure was used: after ILM peeling, no extra flap manipulation was performed. The macular hole was covered by the inverted ILM flap because of the air pressure at the time of the fluid-air exchange. RESULTS: At 12 months, macular hole closure was observed in 40 eyes (97.6%) in Group 1 and in 39 eyes in Group 2 (97.5%). U-shape closure rate, ellipsoid zone defects, and external limiting membrane defects were similar in both groups. The results indicate no statistical difference in anatomical and functional success between both groups. CONCLUSION: The macular hole closure rate, improved visual acuity, and no extra complications indicate noninferiority of the modified inverted ILM technique. Internal limiting membrane finishing, tucking, and massage may not be required to obtain surgical success.

INVERTED INTERNAL LIMITING MEMBRANE FLAP TECHNIQUE VERSUS COMPLETE INTERNAL LIMITING MEMBRANE REMOVAL IN MYOPIC MACULAR HOLE SURGERY: A Comparative Study.

PURPOSE: To compare the results of vitrectomy with complete internal limiting membrane (ILM) removal and inverted ILM flap in the treatment of myopic macular hole (MMH). METHODS: Seventy eyes of 68 patients with MMH undergone pars-plana vitrectomy (PPV) with either complete ILM removal (n = 36, Group 1) or inverted ILM flap technique (n = 34, Group 2) were included in the study. Outcomes measured were the rate of MMH closure assessed by optical coherence tomography (OCT) and visual acuity (BCVA) at six months. RESULTS: Closure of MMH was achieved in 22 cases of Group 1 (61%) and in 32 cases of Group 2 (94%). Surgical failure was reported in 14 cases of Group 1 (39%) and in one case of Group 2 (3%). Average best-corrected visual acuity (BCVA) changed from 0.60 to 0.58 in Group 1 (P = 0.329) and from 0.70 to 0.39 in Group 2 (P < 0.01). Logistic regression analysis showed that inverted ILM flap technique was associated with 22 times higher probability of anatomic success, regardless of the MMH diameter. CONCLUSION: Inverted ILM flap should be preferred to complete ILM removal for the treatment of MMH. The outcomes reported with this technique were better than any other technique described until now.

Internal limiting membrane transplantation for unclosed and large macular holes.

BACKGROUND: To present the surgical technique and clinical outcomes of transplantation of autologous internal limiting membrane (ILM) for large macular holes (MHs) after failed surgeries with ILM removal. METHODS: Thirteen eyes of 13 consecutive patients with MHs larger than 500 µm after failed surgeries with ILM removal underwent vitrectomy with transplantation of autologous ILM. In the ILM transplantation technique, a small piece of the ILM was peeled off and transplanted inside the macular hole. Fluid-air exchange was then performed. The air was then replaced with 10 % perfluoropropane (C3F8) gas. Comprehensive ophthalmologic examinations and spectral-domain optical coherence tomography were performed preoperatively and postoperatively. The main outcome measures were best-corrected Snellen visual acuity (BCVA) and MH closure rate. RESULTS: The preoperative mean base diameter of the MHs was 1637.6 + 412.7 µm (range, 814-2092 µm). The preoperative mean minimum diameter was 814.4 + 255.0 µm (range, 546 µm-1485 µm). Complete MH sealing was achieved in 12 eyes after transplantation of the ILM flap. The mean BCVA was 1.15 + 0.21 (range, 1.0-1.6) before surgery and 0.99 + 0.17 (range, 0.7-1.3) at 12 months postoperatively. There was a significant difference in BCVA before versus after the surgery (t = 3.825, P = 0.0002, paired t- test). CONCLUSIONS: Transplantation of autologous ILM is an effective addition to the surgical options for large macular holes after failed surgeries with ILM removal.

Autologous Internal Limiting Membrane Fragment Transplantation for Large, Chronic, and Refractory Macular Holes.

OBJECTIVE: To evaluate a technique of autologous internal limiting membrane (ILM) fragment transplantation for the treatment of large, chronic, and/or refractory macular holes (MH). DESIGN: This was a 6-month prospective interventional case series. METHOD: Ten eyes of 10 patients with MH underwent pars plana vitretomy (PPV) and ILM peeling followed by transplantation of an autologous ILM fragment to the MH. Six patients had primary MH with an internal diameter greater than 500 µm and a duration of more than 18 months, including 1 patient with nonproliferative diabetic retinopathy previously treated with panretinal photocoagulation. Four eyes with MH had previously been submitted to PPV (i.e. 1 for retinal detachment and 3 to attempt to close large MH). One of the latter also displayed juxtapapillary choroidal neovascularization due to age-related macular degeneration. The primary and secondary outcomes were MH closure and improvement of the best corrected visual acuity (BCVA), respectively. RESULTS: Complete MH closure was achieved in all cases. A statistically significant improvement in the average BCVA was observed after 6 months of follow-up (p = 0.018; paired t test). The BCVA improved in 8 eyes (80%), and in 6 of those eyes it improved by ≥ 15 letters. In 1 patient, the BCVA remained unchanged after the surgery, but the visual field reportedly improved. One patient experienced a slight worsening (0.16 logMAR). Two cases developed atrophy of the retinal pigment epithelium despite MH closure and BCVA improvement. CONCLUSION: Treatment with autologous ILM fragment transplantation seems to be an efficient alternative for large, chronic, and refractory MH.

Autologous internal limiting membrane transplant for recurrent idiopathic macular holes.

PURPOSE: To report a series of recurrent idiopathic macular holes treated by means of a free autologous internal limiting membrane flap and compare visual and anatomic results to a control group undergoing further internal limiting membrane peeling and novel gas tamponade. METHODS: Retrospective surgical series of 15 consecutive patients receiving autologous internal limiting membrane flap compared to 14 patients operated on for internal limiting membrane peeling enlargement. Autologous internal limiting membrane flap was created after brilliant blue G staining, internal limiting membrane lifting, perfluorocarbon bubble injection and creation of a wide internal limiting membrane free flap translocated underneath perfluorocarbon liquid, to the macular hole bed. Both groups were tamponated with 20% SF6 and positioned face down for 4 h a day for 3 days. RESULTS: Macular hole closed in 14/15 (93.3%) patients of the autologous internal limiting membrane group and 9/14 (64.2%) controls (p < 0.05). Visual acuity increased from 0.05 ± 0.03 to 0.23 ± 0.13 Snellen in the autologous internal limiting membrane group and from 0.05 ± 0.03 to 0.14 ± 0.10 Snellen of controls (p < 0.05 for both). Vision of the autologous internal limiting membrane group improved more than controls at 1 month (p = 0.043) and 3 months (p = 0.045). Inner segment/outer segment interruption at 3 months was smaller in the autologous internal limiting membrane group than controls, reducing from 1230 ± 288 µm at baseline to 611 ± 245 and 547 ± 204 µm at 3 months versus 1196 ± 362, 745 ± 222 and 705 ± 223 µm, respectively (p < 0.05). CONCLUSION: Autologous internal limiting membrane flap can effectively close recurrent idiopathic macular holes with a higher closure rate, smaller residual inner segment/outer segment line interruption and higher visual acuity at 3 months than previous standard of care.

Autologous blood pleurodesis in rats to elucidate the amounts of blood required for reliable and reproducible results.

BACKGROUND: Pleurodesis is used in the treatment of spontaneous pneumothorax or refractory pleural effusions of different etiologies. Several agents have been employed, but many questions remain unanswered about their effectiveness and toxicity. Use of autologous blood pleurodesis in clinical practice has been described in the literature without any clear consensus regarding its efficacy. Experimental studies using this technique are limited to a single study in rabbits. We performed a prospective, randomized, observer-blinded, controlled study to evaluate the safety and efficacy of increasing doses of autologous blood pleurodesis in a novel rat model. MATERIALS AND METHODS: Twenty-eight albino Wistar rats were divided into four groups. Groups 1, 2, and 3 were the study groups and group 4 was the control group, with seven animals in each group. Groups 1, 2, and 3 were given autologous blood, 1 mL/kg, 2 mL/kg, 3 mL/kg, respectively, and group 4 (control) was given only 2 mL/kg saline intrapleurally. The rats were sacrificed on postoperative day 30. The surfaces were graded by macroscopic (visible adhesion formation) and microscopic (inflammation and fibrosis) examination. RESULTS: Macroscopically, group 2 and group 3 developed significantly more adhesions; 3 mL/kg autologous blood produced the most significant pleurodesis with generalized adhesions seen between visceral, parietal, and mediastinal pleura. Microscopic examination showed that all study groups developed an inflammatory response at the site of blood injection. There were no pathologic changes in ipsilateral and contralateral lung parenchyma. CONCLUSIONS: Autologous blood at doses 2-3 mL/kg were shown to be effective to produce adhesions in 30 d, and the results were highly reproducible in all rats. We propose that the occasional negative results obtained in humans may be related to an insufficient amount of injected blood, as observed in our rat model.

Internal Limiting Membrane Transposition for Persistent Macular Holes Using Double Layers of Viscoelastic.

Internal limiting membrane (ILM) grafting provides a useful option for repair of large and refractory macular holes that fail to close following prior ILM removal. However, current ILM graft techniques are associated with several challenges that may result in failure, most notably the difficulty in maintaining the graft in situ. In this video, the authors describe their modified technique for ILM grafting using a double layer of viscoelastic for stabilization in situ during the procedure. Four of five eyes managed with this technique demonstrated type 1 closure, and all eyes demonstrated improvement in visual acuity.