Enzymatic vitreolysis using reengineered Vibrio mimicus-derived collagenase.

Purpose: Collagen is a key player contributing to vitreoelasticity and vitreoretinal adhesions. Molecular reorganization causes spontaneous weakening of these adhesions with age, resulting in the separation of the posterior hyaloid membrane (PHM) from the retina in what is called complete posterior vitreous detachment (PVD). Incomplete separation of the posterior hyaloid or tight adherence or both can lead to retinal detachment, vitreomacular traction syndrome, or epiretinal membrane formation, which requires surgical intervention. Pharmacological vitrectomy has the potential of avoiding surgical vitrectomy; it is also useful as an adjunct during retinal surgery to induce PVD. Previously studied enzymatic reagents, such as collagenase derived from Clostridium histolyticum, are nonspecific and potentially toxic. We studied a novel collagenase from Vibrio mimicus (VMC) which remains active (VMA), even after deletion of 51 C-terminal amino acids. To limit the activity of VMA to the vitreous cavity, a fusion construct (inhibitor of hyaluronic acid-VMA [iHA-VMA]) was made in which a 12-mer peptide (iHA, which binds to HA) was fused to the N-terminus of VMA. The construct was evaluated in the context of PVD. Methods: VMA and iHA-VMA were expressed in Escherichia coli, purified, and characterized with gelatin zymography, collagen degradation assay, fluorescamine-based assay, and cell-based assays. Two sets of experiments were performed in New Zealand albino rabbits. Group A (n = 10) received iHA-VMA, while group B (n = 5) received the equivalent dose of VMA. In both groups, saline was injected as a control in the contralateral eyes. Animals were monitored with indirect ophthalmoscopy, optical coherence tomography (OCT), and B-scan ultrasonography. Retinal toxicity was assessed with hematoxylin and eosin (H&E) staining of retinal tissue. Results: The activity of iHA-VMA and VMA was comparable and 65-fold lower than that of C. histolyticum collagenase Type IV. In the iHA-VMA group, all the rabbits (n = 10) developed PVD, with complete PVD seen in six animals. No statistically significant histomorphological changes were seen. In the VMA group, four of the five rabbits developed complete PVD; however, retinal morphological changes were seen in two animals. Conclusions: iHA-VMA displays targeted action confined to the vitreous and shows potential for safe pharmacologic vitreolysis.

Non-reversible tissue fixation retains extracellular vesicles for in situ imaging.

Extracellular vesicles (EVs) are secreted nanosized particles with many biological functions and pathological associations. The inability to image EVs in fixed tissues has been a major limitation to understanding their role in healthy and diseased tissue microenvironments. Here, we show that crosslinking mammalian tissues with formaldehyde results in significant EV loss, which can be prevented by additional fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for visualization of EVs in a range of normal and cancer tissues.

Bush-like integrin filament networks associated with hyaloid vasculature in murine neonate eyes.

The vitreous of perinatal mice temporarily develops a unique vascular system, called the vasa hyaloidea propria (VHP). Observations showed the vessels possessed an extracellular matrix including the basement membrane in their entire length. Immunostaining of whole mount preparations of VHP with integrin ß1 antibody displayed a bush-like network consisting of long and straight fibers which were associated with the VHP but extended apart from the blood vessels. Electron microscopically, each fiber was composed of a bundle of thin filaments different from collagen fibrils. Macrophages associated with the VHP appeared to be arrested by the integrin bushes. The integrin bushes fragmented and disappeared by postnatal day 10, just before the regression of the VHP. Macrophages were involved in the digestion and clearance of integrin bushes. The vitreous integrin bushes appear to provide a scaffold for architectural maintenance of the hyaloid vessels and macrophages.

[The vitreous and the macula].

The macula is a site where various vitreoretinal disorders occur. In 1983 we started to observe the retinal surface of postmortem eyes with a scanning electron microscope (SEM). We investigated the anatomy of the vitreous in postmortem eyes by slit lamp biomicroscopy. The novel anatomy of the premacular vitreous led us to conduct a clinical study of vitreomacular interface diseases. In 1997, time domain optical coherence tomography(OCT) became available which facilitated visualization of the vitreoretinal interface. Swept source OCT which was introduced in 2012 can depict liquefied lacunae in the vitreous. It enabled us to elucidate the mechanism of vitreoretinal diseases. I. SEM revealed the remnants of vitreous cortex at fovea with high incidence (44%), which suggests strong vitreoretinal attachment at the fovea and vitreous cortex origin of the epiretinal membrane. II. We studied the anatomy of the vitreous in postmortem eyes. The vitreous of bisected eye balls was stained by fluorescein and immersed in water and observed by slit-lamp biomicroscopy. We discovered a "posterior precortical vitreous pocket (PPVP)" in adult eyes without posterior vitreous detachment (PVD). III. We performed clinical study in various vitreoretinal diseases based on the novel vitreous anatomy and explained their mechanism. 1. In diabetic retinopathy, ring shaped fibrovascular tissue surrounding the macula is formed along the outer margin of the PPVP. Although PVD progresses outside the PPVP, its posterior wall remains attached to the retina, which causes macular traction or cystoid macular edema. 2. In eyes with idiopathic epimacular membrane (IEM), detached vitreous cortex had an oval defect corresponding to the IEM. Posterior wall of the PPVP that is premacular vitreous cortex appeared to be the framework of IEM. 3. During vitrectomy for macular hole, premacular round defect appears when PVD is created. The residual cortex on the macula is fibrous membrane with elasticity. The tangential contraction of premacular cortex may generate anterior traction to the fovea, which leads to macular hole. IV. Using time domain OCT, we demonstrated the evolution of macular hole, myopic foveoschisis and lamellar macular hole. After 2007, we investigated age related changes of vitreoretinal interface by spectral domain OCT V. We demonstrated whole structure of the PPVP using swept source OCT. PPVP is a boat shaped premacular liquefied space which has a connecting channel to Cloquet's canal. PPVP develops during childhood. Visualization of vitreous structure proved that our previous assumptions are reasonable. Although the physiological function of the PPVP is unclear, we speculate that the aqueous flows into the PPVP though Cloquet's canal and the connecting channel.

Chromovitrectomy and the vitreoretinal interface.

It still remains unclear to which extent the presence and the amount of retinal debris seen in internal limiting membrane (ILM) specimens harvested during macular surgery for macular holes or epiretinal membranes are related to the procedure of ILM peeling itself or to modifications of the surgical technique, such as application of vital dyes for visualization of the ILM, or to pathological conditions with epiretinal membrane formation at the vitreoretinal interface. The presence of cellular fragments on the retinal side of the removed ILM appears to be of multifactorial origin, and additional causes besides dye application need to be considered. However, morphological studies with evaluation of vital dyes are still of relevance and provide additional insights into the ultrastructure of the vitreoretinal interface and its interaction with adjuvants used during macular surgery. Chromovitrectomy is an emerging field in vitreoretinal surgery. It is of importance to better understand the tissue-dye interactions, which not only alter the mechanical properties of the tissue being stained, but may also have an impact on the functional result postoperatively.

Oxidative responses induced by pharmacologic vitreolysis and/or long-term hyperoxia treatment in rat lenses.

PURPOSE: The aim of the study was to investigate the protective effects of intact vitreous gel on the lens after pharmacologic vitreolysis and hyperoxia exposure in rats in vivo. METHODS: Eyes of Sprague-Dawley rats were induced to posterior vitreous detachment (PVD) by pharmacologic vitreolysis, and the rats with and without PVD were treated with hyperoxia 3 h per day for 5 months. Lens transparency was monitored by a slit-lamp biomicroscope. A series of biochemical measurements were made in extracts of the lens cortex and nucleus. Ascorbate levels were measured in the aqueous and vitreous humors. RESULTS: No significant differences in lens transparency or morphology were observed in all groups, and no significant biochemical changes were observed in the cortex or nucleus of lenses of the PVD group. In the lens nucleus, the values of water-soluble protein concentration in PVD + hyperoxia group were lower than that of the PVD group. The levels of water-soluble proteins, glutathione (GSH) and ascorbate decreased in the hyperoxia group with an intact vitreous body. Vitreolysis enhanced the effect of hyperoxia, decreasing soluble protein, GSH and ascorbate below the levels seen in eyes with vitreolysis alone. The levels of antioxidants and soluble proteins were lower in the lens nucleus, and the effects of vitreolysis plus hyperoxia were more significant in the nucleus. Hyperoxia and hyperoxia plus vitreolysis reduced catalase activity and increased oxidized GSH to a greater extent in the lens cortex, although these treatments increased protein-GSH mixed disulfides in both regions. Long-term hyperoxia also lowered ascorbate levels in the vitreous and aqueous humors, an effect that was enhanced by vitreolysis. CONCLUSIONS: Exposure to excess molecular oxygen produces significant oxidative damage to the lens, especially the lens nucleus. These effects were enhanced by pharmacologic vitreolysis, indicating that intact vitreous gel protects the lens from oxidative damage.

Measuring the intravitreal mobility of nanomedicines with single-particle tracking microscopy.

AIM: To develop a robust assay to evaluate and compare the intravitreal mobility of nanoparticles in the intact vitreous body. MATERIALS & METHODS: Excised bovine eyes were prepared to preserve the fragile structure of the vitreous humor, while permitting high-resolution fluorescence microscopy and single-particle tracking analysis of intravitreally injected nanoparticles. This assay was validated by analyzing polystyrene beads and further employed to evaluate gene nanomedicines composed of poly(amido amine)s and plasmid DNA. RESULTS: The assay was able to distinguish immobilized cationic nanoparticles from mobile PEGylated nanoparticles. PEGylation of the polyplexes resulted in a drastic improvement of their mobility. CONCLUSION: An ex vivo eye model is presented for studying nanoparticle mobility in intact vitreous humor by single-particle tracking microscopy. These results give important guidelines for developing gene- and drug-delivery nanomedicines that are compatible with intravitreal administration.

Assessment of vitreous incarceration in sclerotomies with OCT, ultrasound biomicroscopy, and direct visualization.

BACKGROUND AND OBJECTIVE: To compare anterior-segment optical coherence tomography (AS-OCT), ultrasound biomicroscopy (UBM), and direct visualization for detecting vitreous incarceration in sutureless sclerotomies. MATERIALS AND METHODS: Prospective, randomized, and observer-masked experimental study in which 23-gauge vitrectomy was performed in pig eyes. Postoperative incisional vitreous incarceration was evaluated by AS-OCT, UBM, and direct visualization. RESULTS: One hundred eighteen pig eyes were included. Vitreous entrapment was found in 7.9% (28 of 354), 59.6% (211 of 354), and 95.5% (338 of 354) of the sclerotomies analyzed by AS-OCT, UBM, and direct visualization, respectively. Direct visualization was the most sensible method for identifying incisional vitreous when compared with UBM and AS-OCT (P < .0001). In turn, UBM was superior to AS-OCT for observing vitreous incarceration (P < .0001). CONCLUSION: Direct visualization is the most effective method for detecting vitreous entrapment. Regarding the imaging techniques, UBM was superior to AS-OCT for identifying vitreous incarceration.

Use of lutein and zeaxanthin alone or combined with Brilliant Blue to identify intraocular structures intraoperatively.

PURPOSE: To determine whether a natural dye solution based on lutein and zeaxanthin alone or combined with Brilliant Blue stains and facilitates peeling of intraocular membranes in human eyes. METHODS: In this study of 60 cadaveric eyes, open-sky vitrectomy including posterior hyaloid detachment was performed. Different lutein and zeaxanthin concentrations (0.01-20%) were tested alone or combined with different Brilliant Blue concentrations (0.0125-0.025%) in the corneal endothelium, corneal epithelium, anterior and posterior capsule, vitreous cavity through the macula including the posterior hyaloid, and internal limiting membrane. The various dye solutions were in contact with the intraocular membranes for <1 minute and then were removed by mechanical aspiration or membrane peeling initiated and completed with intraocular forceps. The specimens were examined by light and electron transmission microscopy. RESULTS: Contact between lutein and zeaxanthin and the retinal, lens, and vitreous surface resulted in orange and greenish staining of the intraocular membranes, which facilitated surgical steps in all eyes. Lutein and zeaxanthin alone was useful for vitreous identification and lutein and zeaxanthin combined with Brilliant Blue had strong affinity for internal limiting membrane and anterior capsule. Light microscopy confirmed internal limiting membrane removal in all eyes tested. No dye solutions remained in the eyes after the membrane removal. CONCLUSION: A natural dye solution based on lutein and zeaxanthin alone or combined with Brilliant Blue efficiently stained the anterior capsule, vitreous, and internal limiting membrane in human cadaveric eyes and may be a useful tool for vitreoretinal or cataract surgery.

Retinal ectopias and mechanically weakened basement membrane in a mouse model of muscle-eye-brain (MEB) disease congenital muscular dystrophy.

PURPOSE: Some forms of congenital muscular dystrophy are associated with cortical and retinal dysplasias. Protein O-mannose N-acetylglucosaminyltransferase 1 (POMGnT1) knockout mice, one of the mouse models of muscular dystrophy, exhibit a thinner retina with reduced density of retinal ganglion cells. This study is aimed to further characterize the knockout retina, with special emphasis on the inner limiting membrane, the basement membrane of the retina. METHODS: Immunofluorescence staining and transmission electron microscopy were used to analyze the retinas. Atomic force microscopy was performed on the inner limiting membrane preparations to examine their mechanical properties. RESULTS: The inner limiting membrane of the knockout mice exhibited frequent breaks with protrusions of the Müller glial processes and ectopic placement of retinal ganglion cells into the vitreous humor. Disruptions in inner limiting membrane integrity developmentally precede the cellular abnormalities. Regions of disrupted inner limiting membrane were also associated with molecular abnormalities of Müller glia that included diminished presence of the integral membrane proteins Kir4.1 (an inwardly rectifying potassium channel) and aquaporin-4. When measured with atomic force microscopy, the POMGnT1 knockout mouse inner limiting membrane (ILM) exhibited significantly reduced Young's modulus and is therefore mechanically weaker than the ILM from controls. CONCLUSIONS: Deficiency of POMGnT1-mediated glycosylation of dystroglycan is implicated in reduced stiffness of the ILM. The weakened ILM results in the disruption of the membrane and subsequent reduction in retinal integrity.

Possible contribution of hyalocytes to idiopathic epiretinal membrane formation and its contraction.

AIM: To address the cellular components and the contractile mechanisms of the idiopathic epiretinal membrane (ERM). METHODS: Ten surgically removed ERMs were fixed in 4% paraformaldehyde and analysed by whole-mount immunohistochemistry with anti-glial fibrillar acidic protein (GFAP) and alpha smooth-muscle actin (alphaSMA) antibodies. Type I collagen gel contraction assay, an established wound-healing assay in vitro, was performed using cultured bovine hyalocytes or normal human astrocytes (NHA) to evaluate the contractile property of the cells in the presence of tissue growth factor (TGF)-beta2. The expression of alphaSMA was also analysed by western blot analysis to examine myofibroblastic transdifferentiation of the cells. Vitreous-induced collagen gel contraction was also evaluated. RESULTS: All membranes were composed of alphaSMA immunopositive cells in contracted foci and GFAP immunopositive cells in the periphery. No apparent double positive cells were observed in any membranes examined. Cultured hyalocytes showed overexpression of alphaSMA and hypercontraction of collagen gels in response to TGF-beta2, while glial cells showed marginal change. The vitreous from ERM patients also caused overexpression of alphaSMA and hypercontraction of the gels embedding hyalocytes, which were almost completely inhibited in the presence of anti-TGF-beta2 neutralising antibody. CONCLUSIONS: Hyalocytes might be one of the critical components of ERM mediating its contractile property through the effect of TGF-beta2 in the vitreous fluid.

Ultrastructural correlation of spectral-domain optical coherence tomographic findings in vitreomacular traction syndrome.

PURPOSE: To examine the ultrastructural correlates of spectral-domain optical coherence tomography (SD-OCT) findings in patients with vitreomacular traction (VMT). DESIGN: Observational case series. METHODS: Retrospective analysis of six eyes of consecutive patients who underwent vitrectomy surgery for VMT was performed in this single-center, noncomparative study. One patient had a concurrent macular hole. Preoperative assessment included SD-OCT examination with 3-dimensional image reconstruction. During surgery the vitreous cone was dissected from the vitreous body using scissors, then removed from the surface of the retina with a combination of sharp dissection and peeling, and subsequently submitted for histologic and transmission electron microscopic processing. RESULTS: SD-OCT showed prominent vitreal-foveal adhesion in all six eyes. Each eye had an epiretinal membrane (ERM) under the detached perifoveal posterior vitreous detachment. In all eyes this ERM appeared to course up the cone of attached vitreous and along the back surface of the posterior vitreous face. Ultrastructural analysis showed fibrocellular proliferations in the vitreous specimens in all six cases, which included retinal pigment epithelium (RPE) cells (five eyes), fibrocytes (four eyes), and macrophages (three eyes). CONCLUSIONS: The adhesion between the vitreous and fovea in vitreomacular traction syndrome is accompanied by fibrocellular proliferation along the exposed surfaces of the inner retina and the posterior surface of the vitreous. This fibrocellular proliferation may augment the adhesion between the vitreous and fovea, and may account for the prominent OCT signal seen along the posterior surface of the vitreous in these cases.

Unique structure and optics of the lesser eyes of the box jellyfish Tripedalia cystophora.

The visual system of box jellyfish comprises a total of 24 eyes. These are of four types and each probably has a special function. To investigate this hypothesis the morphology and optics of the lesser eyes, the pit and slit eyes, were examined. The pit eyes hold one cell type only and are probably mere light meters. The slit eyes, comprising four cell types, are complex and highly asymmetric. They also hold a lens-like structure, but its optical power is minute. Optical modeling suggests spatial resolution, but only in one plane. These unique and intriguing traits support strong peripheral filtering.

A new strategy to replace the natural vitreous by a novel capsular artificial vitreous body with pressure-control valve.

PURPOSE: The current vitreous substitutes such as silicone oil, heavy silicone oil, and polymeric gels that are directly injected into vitreous cavity frequently cause severe intraocular complications. There is a very urgent need to find a more suitable artificial vitreous substitute for pars plana vitrectomy (PPV) surgery. METHODS: We have devised a novel capsular artificial vitreous using tailor-made silicone rubber elastomer. The novel device was implanted into the vitreous cavity of rabbit after PPV and the eye was examined by ophthalmoscopy, fundus photography, and tonometry during an 8-week treatment period. B-scan ultrasonography, electroretinogram (ERG), and histological studies by light microscopy were also performed at the end of 8 weeks. RESULTS: The novel artificial vitreous body consists of a thin vitreous-like capsule with a silicone tube-valve system. The capsule can be folded and implanted into vitreous cavity through 1.5 mm incision on sclera. Physiological balanced solution (PBS) was then injected into the capsule and inflated to support retina and control intraocular pressure (IOP) through the tube-valve system subsequently fixed under the conjunctiva. Experiments using rabbits showed that the novel vitreous body could effectively support the retina and apparently induced no significant pathological changes in the eye over 8 weeks. CONCLUSION: This approach may provide a new research strategy in the vitreous replacement technology. The novel artificial vitreous body device can effectively support retina, control IOP, and has good biocompatibility. It may be a good alternative to injecting artificial vitreous although its tamponade properties and usefulness still have to be proven in complex vitreoretinal diseases.

[The primary objective in macular hole surgery. Ultrastructural features of the vitreomacular interface]. / Operative Zielvorgabe in der Makulaforamenchirurgie. Neue Aspekte zur Feinstruktur der vitreomakulären Grenzfläche.

We compared the ultrastructure of the inner limiting membrane (ILM) and epiretinal tissue in closed and non-closed, idiopathic macular holes (MH). Peeling of ILM and epimacular tissue during vitrectomy was successfully performed on 77 eyes with stage III MH and on 19 eyes with stage IV MH. In 16 additional eyes with non-closed MH, we performed a second vitrectomy with extended ILM removal. The specimens were processed for transmission electron microscopy. Fibrocellular proliferation at the vitreal side of the ILM was found in 57% of MH that were closed by one operation, and in 100% of non-closed MH. Mono- and multilayered cellular membranes as well as native vitreous collagen at the ILM were significantly more frequent in eyes with stage IV MH than in eyes with stage III MH. In non-closed MH, cellular proliferation was mostly seen as irregular cell accumulation, and masses of newly formed collagen were found. Since ILM remnants and collagen represent a stimulus for the early formation of tangential traction preventing successful MH closure, it appears mandatory to create a complete vitreoretinal separation and to remove the ILM and collagen thoroughly during MH surgery.

[Comparative ultrastructural analysis of the proliferative tissue in PDR patients with or without vitreous haemorrhage: from ultrastructural approach to clinical practice]. / Ultrastruktureller Vergleich des proliferativen Gewebes bei PDR-Patientenmit und ohne Glaskörperblutung--von Grundlagenforschungen zu den klinischen Therapien.

BACKGROUND: Proliferative diabetic retinopathy (PDR) is a sight-threatening disease with high social impact, easily complicated with vitreous haemorrhages (VH). In our study we try to point out the ultrastructural differences between proliferative tissues in PDR patients with and without vitreous haemorrhage in regard to the clinical practice and proper timing of vitreo-retinal surgery. MATERIAL AND METHODS: In our prospective study we included 27 PDR patients (18 with PDR only and 9 with PDR and vitreous haemorrhage). All of them were later operated with pars plana vitrectomy, during which proliferative tissue was collected. The materials were examined with transmission and scanning electron microscopy and histochemically with Safranin O. RESULTS: The proliferative tissue of PDR patients without VH was mainly comprised of fibroblasts, macrophages and glial cells. We found a variety of blood vessels. Most common were the capillaries of "mature" new vessels. In the proliferations of patients with VH, macrophages and erythrocytes were more commonly detected. In the tissue capillaries of young new vessels prevailed, with only a very thin layer of endothelial cells comprising their walls. In the extracellular matrix among the proteoglycan complexes, we found lots of residual elements of blood. CONCLUSIONS: Our results point out that blood in the vitreous cavity alters the ultrastructure of the existing PDR proliferations in respect to making them more rigid and prone to contractions. It is therefore important to consider vitreous haemorrhage in PDR patients as a very serious complication, requiring in some cases urgent vitreo-retinal surgery.

Vitreoretinal surgery using bromphenol blue as a vital stain: evaluation of staining characteristics in humans.

OBJECTIVE: To evaluate the staining characteristics of bromphenol blue used during vitreoretinal surgery in humans. PATIENTS AND METHODS: 13 patients with epiretinal membranes were included. Before and after surgery a complete clinical examination including best corrected visual acuity, funduscopy, fluorescein angiography, OCT (Stratus), Goldmann perimetry and multifocal ERG as well as photography of the macular area was performed. Bromphenol blue was used in concentrations of 0.2% in most patients. Removed epiretinal tissue was evaluated using electron microscopy. RESULTS: Using dye concentrations of 0.2% a good demarcation of epiretinal membranes was seen in 11/13 patients. Staining of vitreous remnants at the vitreous base was seen in all patients. No dye-related adverse events were seen during follow-up in the functional tests (VA, ERG, perimetry) performed. Histological evaluation of epiretinal membranes showed unremarkable aspects of epiretinal cellular layers and unremarkable retinal surface of the internal limiting membrane (ILM). CONCLUSION: Bromphenol blue appears to be a very helpful and safe tool in posterior segment surgery. The staining characteristics need to be further evaluated in prospective study settings and larger numbers of patients.

Long-term retention of dye after indocyanine green-assisted internal limiting membrane peeling.

PURPOSE: To evaluate dye retention in the fundus after indocyanine green (ICG)-assisted internal limiting membrane peeling. METHODS: Ten eyes with stage 3 or 4 nondiabetic idiopathic macular hole (MH group) and six eyes with diffuse diabetic macular edema (DM group) were studied. The fundus was examined with 780-nm infrared illumination by a scanning laser ophthalmoscope (SLO) after ICG-assisted internal limiting membrane peeling. The postoperative follow-up period ranged from 6 to 12 months (mean+/-SD, 3.7+/-2.6 months). RESULTS: Fluorescence from ICG was detected in all studied eyes in both groups up to 6 months after surgery. At 9 months after surgery, ICG fluorescence was visible in all eyes of the DM group, but in only one-third of eyes of the MH group. No fluorescence was detected in fellow eyes that had not been operated on. CONCLUSION: The present study using SLO revealed that ICG remains in the fundus for over 6 months after surgery. The results also suggested that a longer time might be required for dye clearance from the diabetic retina than from the nondiabetic retina.

The macular hole: histopathologic studies.

OBJECTIVE: To delineate the light and electron microscopic features of tissue removed at the time of macular hole surgery. METHODS: The ocular fluid specimens were concentrated using Millipore filters and stained with a modified Papanicolaou and the periodic acid-Schiff stains in 697 cases. In 92 cases, surgically isolated tissue was processed and examined by electron microscopy. RESULTS: The findings in the specimens studied by the Millipore filter technique included vitreous strands; cellular fragments in 108 cases (13.3%); fibrocellular fragments in 75 (9.2%); and fragments of internal limiting lamina (ILL) of the retina in 104 (12.8%). Findings in the 92 specimens with tissue studied by electron microscopy included native vitreous collagen in 48 cases (52.2%); new collagen in 6 (6.5%); native and new collagen in 1 (1.2%); ILL of the retina in 54 (58.7%); and a variety of cells in 22.5% of cases, including fibrocytes, myofibrocytes, fibrous astrocytes with and without myoblastic features, Mueller cells, retinal pigment epithelium with and without myoblastic features, and inflammatory cells. The organization of the tissue elements included a cellular layer along one surface of a layer of cortical vitreous in 18 cases, cortical vitreous along the inner surface of the ILL of the retina in 10, and cortical vitreous sandwiched between the ILL of the retina and a layer of cells in 9. CONCLUSIONS: Tangential traction induced by fluid movements affecting the cortical vitreous is a major factor in the pathogenesis of idiopathic macular holes. Cellular proliferation is a secondary change seen in 22.2% of cases.

Effect of microkeratome suction during LASIK on ocular structures.

PURPOSE: To study the effect of microkeratome suction on ocular structures during LASIK. DESIGN: Observational, prospective case series. PARTICIPANTS: Twenty-one eyes of 11 patients with myopia or astigmatic myopia (8 females, 3 males) were included. The mean patient age was 36.3 years (median, 37 years; range, 24-48 years), and the mean spherical equivalent was -5.03 diopters (D) (median, -4.63 D; range, -2.38 to -8.38 D). METHODS: We performed preoperative and intraoperative A-scan ultrasonography during application of suction using the Hansatome microkeratome (Bausch & Lomb Surgical, Munich, Germany) to create corneal flaps during LASIK. We also performed preoperative and postoperative B-scan ultrasonography of the posterior ocular segment with special attention to the presence and size of posterior vitreous detachment (PVD). MAIN OUTCOME MEASURES: We measured changes in the axial length, anterior chamber depth, lens thickness, and vitreous distance (distance from the posterior lens capsule to the posterior pole) during application of the microkeratome suction ring and recorded new occurrences of or increases in the size of the PVD after surgery. RESULTS: The lens thickness decreased (mean change, -0.20 mm; P = 0.001; 95% confidence interval [CI], -0.11 to -0.30) in 18 eyes during application of the suction ring. The vitreous distance increased (mean change, 0.20 mm; P = 0.004; 95% CI, 0.08-0.32) in 16 eyes. No statistically significant changes were found in the anterior chamber depth (P = 0.75) or axial length (P = 0.51). After surgery, 3 of 14 eyes (21.4%) experienced PVD that did not have echographic signs of PVD before surgery. Of 7 eyes with preoperative PVD, the PVD enlarged in 1 eye (14.3%). CONCLUSIONS: During application of microkeratome suction, the lens thickness decreases, whereas the vitreous distance increases, suggesting anterior traction on the posterior segment. The relationship between the observed PVD and LASIK merits further investigation.