- Email: [email protected]
In Vivo Confocal Microscopy Study of Blebs after Filtering Surgery
In Vivo Confocal Microscopy Study of Blebs after Filtering Surgery Antoine Labbé, MD,1,2 Bénédicte Dupas, MS,1 Pascale Hamard, MD, PhD,1 Christophe Baudouin, MD, PhD1,2,3 Objective: To analyze bleb structure after filtering surgery at the cellular level using a new generation in vivo confocal microscope. Design: Observational case series. Participants: We retrospectively evaluated 17 filtering blebs of 13 patients after trabeculectomy. Methods: Ophthalmologic examinations included slit-lamp examination, applanation tonometry, and in vivo confocal microscopy (Heidelberg Retina Tomograph II, Rostock Cornea Module). Eyes were classified into 3 groups: (1) functioning blebs (6 eyes), (2) nonfunctioning blebs (6 eyes), and (3) functioning blebs after application of mitomycin C (5 eyes). Cellular patterns, morphologic appearance, and functional aspects of functioning and nonfunctioning blebs were compared in a masked manner. Main Outcome Measures: In vivo confocal microscopy images were analyzed for number of intraepithelial microcysts, density of subepithelial connective tissue, presence of blood vessels, or encapsulation. Results: All functioning blebs had numerous intraepithelial optically-empty microcysts, whereas all nonfunctioning blebs had none or few. Subepithelial connective tissue was widely spaced in all functioning blebs, whereas the tissue was dense in 83.3% of nonfunctioning blebs. Functioning blebs with mitomycin C had numerous microcysts and loosely arranged subepithelial connective tissue as compared with nonfunctioning blebs. Conclusions: In vivo confocal microscopy study of blebs is an original method that agrees well with ex vivo histologic examination. The number of microcysts and the density of the subepithelial connective tissue observed with in vivo confocal microscopy are correlated with bleb function. By providing details of the structures of filtering blebs at the cellular level, in vivo confocal microscopy constitutes a new promising way to understand wound healing mechanisms after filtering surgery. Ophthalmology 2005;112:1979 –1986 © 2005 by the American Academy of Ophthalmology.
Trabeculectomy has become the method of choice in surgical treatment of patients with glaucoma.1 This surgical technique is usually associated with an elevation of the conjunctiva overlying the scleral flap (i.e., the filtering bleb). The long-term success of filtering surgery is not only dependent on surgical technique; age, type of glaucoma,2 ethnic origin, prior failed filtering surgery or long-term use of topical medications before surgery3 are also critical factors. However, the development of a filtering bleb, determined by the postoperative wound healing process, is a major factor of efficiency and long-term success of surgical procedures. For these reasons, many authors have investigated Originally received: January 27, 2005. Accepted: May 29, 2005.
Manuscript no. 2005-88.
1
Department of Ophthalmology III, Quinze-Vingts National Ophthalmology Hospital, Paris, France.
2
INSERM U598, University of Paris 5, Paris, France. Department of Ophthalmology, Ambroise Paré Hospital, APHP, University of Versailles, Versailles, France. Supported by Quinze-Vingts National Ophthalmology Hospital, Paris, France. Correspondence to Christophe Baudouin, MD, PhD, Service d’Ophtalmologie III, C.H.N.O. des Quinze-Vingts, 28 rue de Charenton, Paris, 75012 France. E-mail: [email protected]. 3
© 2005 by the American Academy of Ophthalmology Published by Elsevier Inc.
morphologic criteria of these blebs to correlate clinical and functional aspects.4,5 However, in some cases, there is no correlation between bleb appearance or shape and intraocular pressure. The reasons for bleb failure, related to bleb scarring and histological changes within the bleb tissue, are not easy to study in clinical practice. In vivo confocal microscopy can provide details of ocular structures at the cellular level, and it has already been used in numerous studies of normal and pathologic corneas. Recent advances in the in vivo confocal microscopy technique have permitted visualization of peripheral ocular structures. However, in vivo confocal microscopy is a relatively new method for investigating conjunctiva.6 In a preliminary report, we have presented the technique allowing the description of blebs using in vivo confocal microscopy.7 The aim of the present study was to quantify and to compare, in a masked manner the in vivo confocal microscopy aspects of functioning and nonfunctioning blebs, using the new confocal microscope Heidelberg Retina Tomograph II/Rostock Cornea Module (Heidelberg Engineering GmbH, Heidelberg, Germany) to provide a better understanding of filtering mechanisms and the wound healing process after filtering surgery. ISSN 0161-6420/05/$–see front matter doi:10.1016/j.ophtha.2005.05.021
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Patients and Methods In this pilot study, we retrospectively evaluated 17 filtering blebs of 13 patients who had previously undergone trabeculectomy, and who were followed-up at the Quinze-Vingts National Ophthalmology Hospital, Paris, France. Six of the patients were female (46.1%) and 7 were male (53.9%), whose ages ranged from 21 to 79 (mean⫾standard deviation; 55.5⫾16.4 years). There were 10 right eyes (58.9%) and 7 left eyes (41.1%). Of the 17 eyes, 15 (88.2%) had primary open-angle glaucoma and 2 (11.8%) had juvenile open-angle glaucoma. In 5 cases (29.4%), mitomycin C (MMC) had been intraoperatively applied for 2 minutes using a small piece of surgical sponge soaked in 0.2 mg/ml MMC. After exposure, the conjunctival flap area was immediately irrigated with 250 ml balanced salt solution (BSS Alcon, Fort Worth, TX). The period between surgery and in vivo confocal microscopy evaluation ranged from 1 to 228 months (mean⫾standard deviation; 47.9⫾63 months). First, all patients had a slit-lamp examination and Goldmann applanation tonometry. This study was performed in compliance with French regulations on biomedical research, and all individual patients were informed of the aims of recording these data, and their consent was obtained. Patients were examined using a new in vivo confocal microscope (i.e., the Heidelberg Retina Tomograph (HRT) II/Rostock Cornea Module [Heidelberg Engineering GmbH, Heidelberg, Germany]). The HRT II is a confocal scanning laser ophthalmoscope that was initially developed for the analysis of the posterior pole of the eye, especially the optic nerve head, and it has become a standard for investigating glaucomatous changes of the optic disc.8,9 With the addition of the Rostock Cornea Module, the HRT II is converted to an in vivo confocal microscope available for investigating the ocular surface.10 Before the in vivo confocal microscopy examination, 1 drop of topical anesthetic (Novesine 0,4% [oxybuprocaïne 0.4 %] MSD-Chibret, Paris, France) and 1 drop of gel tear substitute (Lacrigel, carbomer 0,2%, Europhta, Monaco) are instilled into the lower conjunctival fornix. The patient is then seated at an examination table with the head into the headrest. The patient fixates with the contralateral eye at a small bright red light. The adjustment of the eye is performed by means of the live image and under control of a charged-coupled device color camera (640 ⫻ 480 pixels, RGB, 15 frames/s). The x-y position of the image and the section depth are controlled manually. The objective of the microscope is an immersion lens (Olympus, Hamburg, Germany), magnification ⫻60, covered by a polymethyl metacrylate cap. The laser source used in the HRT II/ Rostock Cornea Module is a diode laser with a wavelength of 670 nm. Images consist of 384 ⫻ 384 pixels covering an area of 400 m ⫻ 400 m, with transversal optical resolution of 2 m and longitudinal optical resolution of 4 m (Heidelberg Engineering). For all eyes, several confocal microscopic images of the superficial epithelium and of the subepithelial connective tissue of the conjunctiva located over the trabeculectomy site were taken. Each eye was examined for less than 5 minutes and no complications related to in vivo confocal microscopy evaluation were noted. Eyes were divided into 3 groups: (1) functioning blebs (6 eyes; 35.3%), (2) nonfunctioning blebs (6 eyes; 35.3%), and (3) functioning blebs after MMC application (5 eyes; 29.4%). In the nonfunctioning bleb group (group 2), 2 clinical appearances were analyzed: (1) flat blebs (5 blebs) and (2)encapsulated blebs (1 bleb). Surgical success of filtering surgery was defined by the intraoc-
ular pressure (IOP), according to previous studies.5,11,12 Functioning blebs were defined as IOP ⬍21 mmHg without antiglaucoma treatment, and nonfunctioning blebs were defined as IOP ⱖ21 mmHg and/or the necessity for an antiglaucoma medication. To ensure consistency, images were analyzed retrospectively by a single researcher (CB) who was masked toward clinical features and IOP control. In vivo confocal microscopy images were evaluated for conjunctival and corneal epithelium changes, presence and number of intraepithelial microcysts rated from 0 (none) to 3 (numerous) (Fig 1A), size of microcysts classified as ⬍100 m and ⬎100 m, density of subepithelial connective tissue rated from 0 (loosely) to 3 (dense) (Fig 1B), presence of blood vessels, or encapsulation of the bleb. Statistical values of number of microcysts and density of subepithelial connective tissue (mean⫾standard error of the mean (SEM) were calculated in each group and compared using the nonparametric Mann–Whitney U test. Probability values less than 0.05 were considered significant.
Results For group 1, the functioning blebs in vivo confocal microscopy showed several distinct characteristics. Conjunctival and corneal epithelial cells were perfectly seen and had normal appearance compared with normal eyes13 or patterns found in areas distant from the blebs (Fig 2 [available at http://aaojournal.org]). Between normal epithelial cells, numerous optically clear spaces filled with fluid were seen corresponding to microcysts as observed with the slit lamp (Fig 3A). These microcysts were particularly numerous in functioning blebs, with all the functioning blebs having a number of microcysts rated 2 or 3 (mean⫾SEM, 2.5⫾0.22; P ⫽ 0.0001, as compared with nonfunctioning blebs, mean⫾SEM, 0.667⫾0.21), and were predominantly found in the conjunctiva adjacent to the limbus. The size of the microcysts was between 10 and 150 m in 83.3% of the functioning blebs. Subepithelial connective tissue images showed a loosely arranged tissue, with all the functioning blebs having a density of connective tissue rated 0 or 1 (mean⫾SEM, 0.667⫾0.21; P ⫽ 0.001, as compared with nonfunctioning blebs, mean⫾SEM, 2.5⫾0.34). This tissue was widely spaced and contained clear spaces (Fig 3B). No blood vessel was clearly seen in the subepithelial connective tissue. Regarding group 2, that of the nonfunctioning blebs, conjunctival and corneal cells were again perfectly seen with normal appearance. Very few optical clear spaces corresponding to microcysts were observed between superficial conjunctival cells (mean⫾SEM, 0.667⫾0.21; P ⫽ 0.0001, as compared with functioning blebs, mean⫾SEM, 2.5⫾0.22), and all the nonfunctioning blebs had microcysts rated 0 or 1 (Fig 4A). Three (50%) of the nonfunctioning blebs showed few microcysts containing optically dense material (Fig 4B). Within the clinically encapsulated nonfunctioning bleb, dense fibrotic tissue evocative of encapsulation was observed (Fig 4C). Conversely, an encapsulation-like pattern was observed with in vivo confocal microscopy in 1 clinically flat nonfunctioning bleb. In an encapsulated bleb, microcysts were observed mostly in the peripheral area of the bleb. The subepithelial tissue of nonfunctioning blebs showed a dense, connective tissue with few or no clear spaces, with 83.3% of these blebs having a density that was rated 2 or 3 (mean⫾SEM, 2.5⫾0.34;, P ⫽ 0.001, as compared with functioning blebs, mean⫾SEM,
™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™3 Figure 1. In vivo confocal microscopy images of blebs (400 m⫻400 m). A, Rating of microcysts in the conjunctival epithelium: 0, 1, 2, or 3. B, Rating of density of subepithelial connective tissue: 0, 1, 2, or 3.
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Figure 3. In vivo confocal microscopy images of functioning blebs (400 m⫻400 m). A–D, Examples of numerous microcysts in the conjunctival epithelium. E–H, Examples of subepithelial connective tissue widely spaced, containing clear spaces.
0.667⫾0.21) (Fig 4D). Blood vessels were seen in the subepithelial connective tissue of 4 (66.7%) nonfunctioning blebs (Fig 4D). Regarding group 3, the functioning blebs with MMC, we observed numerous clear spaces corresponding to large confluent microcysts (Fig 5A) between morphologically normal conjunctival epithelial cells. Four of 5 of these blebs had a number of microcysts rated 2 or 3 (mean⫾SEM, 2.2⫾0.22; P ⫽ 0.001, as compared with nonfunctioning blebs, mean⫾SEM, 0.667⫾0.21). These microcysts had different sizes, ranging from 10 to 300 m. Some of these microcysts showed hyper-reflective microdots in the superficial epithelium layer (Fig 5A) in 3 blebs. The nature of these hyper-reflective dots remains unknown, but they might have been necrotic epithelial cells or inflammatory cells. The subepithelial tissue was characterized by loosely arranged connective tissue
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with numerous large clear spaces (Fig 5B), and 4 of 5 of these blebs had a density of subepithelial tissue rated 0 or 1 (mean⫾SEM, 0.8⫾0.37; P ⫽ 0.0084, as compared with nonfunctioning blebs, mean⫾SEM, 2.5⫾0.34). In one of these blebs, an encapsulation was observed with in vivo confocal microscopy. No blood vessel was observed in the subepithelial tissue of functioning blebs with MMC. These results and bleb confocal microscopy image characteristics are shown in Table 1.
Discussion The long-term success of trabeculectomy is mainly dependent on the development of a functioning bleb. The forma-
Labbé et al 䡠 In Vivo Confocal Microscopy and Filtering Blebs
Figure 4. In vivo confocal microscopy images of nonfunctioning blebs (400 m⫻400 m). A–C, Examples of blebs having none or few microcysts in the conjunctival epithelium. D, E, Examples of microcysts containing optically dense material. F, Encapsulation observed at the periphery of the bleb. G, H, Examples of dense subepithelial connective tissue. I, Blood vessels in the subepithelial tissue.
tion and the maintenance of this functioning bleb, with regard to wound healing and conjunctival scarring, are therefore of primary importance. For these reasons, many authors have presented classifications of these blebs to correlate the morphologic criteria observed biomicroscopically with the outcome of these blebs. Picht and Grehn11,12 classified the developing, filtering bleb, showing that favorable bleb development was characterized by microcysts of the conjunctiva, paucity of vessels, diffuse bleb, and moderate elevation of the bleb. In contrast they observed that unfavorable bleb development was characterized by increased vascularization, cork screw vessels, encapsulation
of the bleb and high-domed appearance. Because in some cases the appearance of the bleb is not correlated to IOP, and because the reason of failure is often unclear, some authors have looked for new in vivo evaluation techniques such as ultrasound biomicroscopy14 –16 to understand bleb failure mechanisms. However, ultrasound biomicroscopy evaluation or morphologic criteria observed biomicroscopically can only indirectly observe and cannot analyze histological changes explaining bleb failure. The principle of confocal microscocopy was first described by Minsky in 195717 The technological advances of the past 40 years, have led to the development of in vivo
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Figure 5. In vivo confocal microscopy images of functioning blebs with mitomycin C (MMC) (400 m⫻400 m). A, B, Examples of large, confluent, and numerous microcysts. C, D, Microcysts containing hyper-reflective microdots. E, F, Examples of loosely arranged connective tissue.
confocal microscopy for observation of the human cornea under normal6,18 or pathological conditions.19 –27 More recently, the conjunctival epithelium and stroma could also be observed at the cellular level using a new-generation in vivo confocal microscope.6 In a preliminary report, we described the first patterns of blebs after filtering surgery using this new technique of in vivo confocal microscopy.7 In the present study, we further analyzed these findings more precisely and compared the different types of blebs observed after trabeculectomy. The purpose of this study was to correlate the images obtained with in vivo confocal microscopy, with regard to the number of microcysts, the subepithelial tissue density, and the presence of encapsulation or vessels, with the morphological and functional aspects of these blebs. Functioning blebs (group 1) showed a normal conjunctival epithelium with numerous microcysts and a subepithelial tissue arranged loosely and hypo-reflective, with a high number of optically clear spaces. In contrast, nonfunctioning blebs (group 2) showed none or very few microcysts, and in this case, the subepithelial tissue was hyper-reflective, with dense collagenous connective tissue and blood vessels. For Powers et al,28 who studied functioning blebs using light and electron microscopy, the subepithelial connective tissue was loosely arranged with widely spaced collagen. These authors also described clear spaces in the superficial substantia. In a study of blebs using light and electron
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microscopy, Addicks et al29 showed that nonfunctioning blebs had dense collagenous connective tissue. For these authors, functioning blebs had looser subepithelial connective tissue with histologically clear spaces corresponding to mycrocysts. Our results are in good consistency with ex vivo histological analyses of functioning and nonfunctioning blebs previously described. The presence and number of these microcysts are assumed to be a positive predictive factor for functioning blebs, and interestingly these microcysts were observed in this study as early as the first month after surgery. The morphologic analysis of functioning blebs frequently showed these microcysts.5,11,30 The in vivo confocal microscopic examination of functioning blebs seems to add strength to the argument that these microcysts are channels for the passage of aqueous humor.30 It has been well known for some time that the subepithelial connective tissue of the blebs has a major influence on IOP control. Fibrotic scarring in the subconjunctival space and excessive extracellular matrix production has been previously observed in nonfunctioning blebs.29 –31 In this study, in vivo confocal microscopy images of functioning blebs showing loosely arranged connective tissue and nonfunctioning blebs showing dense subepithelial tissue were well consistent with previous histological studies.28,29 Mitomycin C is applied during filtering surgery to reduce the risk of bleb failure.32 By inhibiting cell proliferation, MMC prevents an excessive healing response
Labbé et al 䡠 In Vivo Confocal Microscopy and Filtering Blebs Table 1. Individual Data and In Vivo Confocal Microscopy Characteristics In Vivo Confocal Microscopy Evaluation Microcysts No.
Eye
Follow-up Period (mos)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
R R L R R R R L L L L R R R L R L
15 228 60 5 96 27 30 28 6 120 144 43 6 2 2 1 1
Connective tissue
Classification of Blebs
Quantity (0 –3)
Size*
Characteristics of Microcysts
Density (0 –3)
Encapsulation
Vessels
F F F F F F Non F, En Non F Non F Non F Non F Non F F with MMC F with MMC F with MMC F with MMC F with MMC
3 3 2 2 2 3 1 1 1 0 0 1 3 2 3 1 2
⬍ ⬍ ⬍ ⬍ ⬍ ⬎ ⬍ ⬍ ⬍ — — ⬍ ⬎ ⬎ ⬎ ⬍ ⬍
E E E E E E E D D — — D E, HMD E, HMD E, HMD E E
1 1 1 1 0 0 3 1 2 3 3 3 0 1 0 2 1
N N N N N N Y N N N Y N N N N Y N
N N N N N N N N Y Y Y Y N N N N N
*Size of microcysts: ⬍100 m; ⬎100 m. D ⫽ dense; E ⫽ empty; F ⫽ functioning bleb; F with MMC ⫽ functioning bleb with mitomycin C; HMD ⫽ Hyper-reflective microdots; L ⫽ left; N ⫽ no; Non F ⫽ nonfunctioning bleb; Non F, En ⫽ nonfunctioning clinically encapsulated bleb, R ⫽ right; Y ⫽ yes.
and scarring, and thus enhances the success of the procedure. Shields et al33 have used light and electron microscopy to analyze an excised bleb after trabeculectomy with MMC; they found numerous microcysts within the epithelium and a loosely arranged connective tissue within the subepithelium. Necrotic nuclei and cytoplasmic vacuoles were also observed in the epithelium of these blebs using electron microscopy.29 The hyper-reflective microdots observed with in vivo confocal microscopy between and within microcysts would likely correspond to these necrotic nuclei. In our study, in vivo confocal microscopy images of functioning blebs with MMC well agreed with previous ex vivo analyses. In vivo confocal microscopy evaluation of blebs after filtering surgery thus allowed us to study, at a cellular level, the conjunctival wall of these blebs. By providing in vivo high-resolution images of these structures, we obtained results similar to ex vivo microscopic analyses. However, we observed a nonfunctioning bleb, according to clinical criteria, with a loosely arranged connective tissue, and one functioning bleb with MMC that had few microcysts. These few cases probably demonstrate the limits of this evaluation. Only a limited part of the conjunctiva covering the bleb is examined at a time, and one cannot make conclusions from such focal examination. Nevertheless, in vivo confocal microscopy evaluation is now providing new insight into the analysis of filtering blebs. This new method is rapid and noninvasive and it allows clinicians and researchers to visualize in vivo functioning or nonfunctioning blebs at the cellular level. Using this technique we can observe directly the histological processes that correlate with filtration or failure. By studying the wound healing mechanisms after filter-
ing surgery in vivo, this technique will permit a better understanding of filtration failure. Clinicians, with images at a cellular level, would be able to predict the outcome of these blebs and eventually provide specific treatments to enhance success rates of their surgical procedures.
References 1. Cairns JE. Trabeculectomy. Preliminary report of a new method. Am J Ophthalmol 1968;66:673–9. 2. Mills KB. Trabeculectomy: a retrospective long-term followup of 444 cases. Br J Ophthalmol 1981;65:790 –5. 3. Broadway DC, Grierson I, O’Brien C, Hitchings RA. Adverse effects of topical antiglaucoma medication. II. The outcome of filtration surgery. Arch Ophthalmol 1994;112:1446 –54. 4. Cantor LB, Mantravadi A, WuDunn D, et al. Morphologic classification of filtering blebs after glaucoma filtration surgery: the Indiana Bleb Appearance Grading Scale. J Glaucoma 2003;12:266 –71. 5. Vesti E. Filtering blebs: follow up of trabeculectomy. Ophthalmic Surg 1993;24:249 –55. 6. Jalbert I, Stapleton F, Papas E, et al. In vivo confocal microscopy of the human cornea. Br J Ophthalmol 2003;87:225–36. 7. Labbe A, Dupas B, Hamard P, Baudouin C. An evaluation of blebs after filtering surgery with the in vivo confocal microscope [in French]. J Fr Ophtalmol 2004;27:1083–9. 8. Medeiros FA, Zangwill LM, Bowd C, Weinreb RN. Comparison of the GDx VCC scanning laser polarimeter, HRT II confocal scanning laser ophthalmoscope, and Stratus OCT optical coherence tomograph for the detection of glaucoma. Arch Ophthalmol 2004;122:827–37. 9. Wollstein G, Garway-Heath DF, Fontana L, Hitchings RA. Identifying early glaucomatous changes. Comparison between expert
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14. 15. 16. 17. 18. 19. 20. 21.
clinical assessment of optic disc photographs and confocal scanning ophthalmoscopy. Ophthalmology 2000;107:2272–7. Stave J, Zinser G, Grummer G, Guthoff R. Modified Heidelberg Retinal Tomograph HRT. Initial results of in vivo presentation of corneal structures [in German]. Ophthalmologe 2002;99:276 – 80. Picht G, Grehn F. Classification of filtering blebs in trabeculectomy: biomicroscopy and functionality. Curr Opin Ophthalmol 1998;9(2):2– 8. Picht G, Grehn F. Development of the filtering bleb after trabeculectomy. Classification, histopathology, wound healing process [in German]. Ophthalmologe 1998;95:W380 –7. Leduc C, Dupas B, Ott-Benoist AC, Baudouin C. Advantages of the in vivo HRT2 corneal confocal microscope for investigation of the ocular surface epithelia [in French]. J Fr Ophtalmol 2004;27:978 – 86. Pavlin CJ, Harasiewicz K, Foster FS. Ultrasound biomicroscopy of anterior segment structures in normal and glaucomatous eyes. Am J Ophthalmol 1992;113:381–9. Avitabile T, Russo V, Uva MG, et al. Ultrasound-biomicroscopic evaluation of filtering blebs after laser suture lysis trabeculectomy. Ophthalmologica 1998;212(Suppl):17–21. Yamamoto T, Sakuma T, Kitazawa Y. An ultrasound biomicroscopic study of filtering blebs after mitomycin C trabeculectomy. Ophthalmology 1995;102:1770 – 6. Minsky M. Memoir on inventing the confocal scanning microscope. Scanning 1988;10:128 –38. Mustonen RK, McDonald MB, Srivannaboon S, et al. Normal human corneal cell populations evaluated by in vivo scanning slit confocal microscopy. Cornea 1998;17:485–92. Masters BR, Bohnke M. Confocal microscopy of the human cornea in vivo. Int Ophthalmol 2001;23:199 –206. Kaufman SC, Musch DC, Belin MW, et al. Confocal microscopy: a report by the American Academy of Ophthalmology. Ophthalmology 2004;111:396 – 406. Chiou AG, Kaufman SC, Beuerman RW, et al. Differential diagnosis of linear corneal images on confocal microscopy. Cornea 1999;18:63– 6.
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22. Pfister DR, Cameron JD, Krachmer JH, Holland EJ. Confocal microscopy findings of Acanthamoeba keratitis. Am J Ophthalmol 1996;121:119 –28. 23. Rosenberg ME, Tervo TM, Muller lJ, et al. In vivo confocal microscopy after herpes keratitis. Cornea 2002;21:265–9. 24. Ciancaglini M, Carpineto P, Zuppardi E, et al. In vivo confocal microscopy of patients with amiodarone-induced keratopathy. Cornea 2001;20:368 –73. 25. Mustonen RK, McDonald MB, Srivannaboon S, et al. In vivo confocal microscopy of Fuch’s endothelial dystrophy. Cornea 1998;17:493–503. 26. Werner LP, Werner L, Dighiero P, et al. Confocal microscopy in Bowman and stromal corneal dystrophies. Ophthalmology 1999;106:1697–704. 27. Pisella PJ, Auzerie O, Bokobza Y, et al. Evaluation of corneal stromal changes in vivo after laser in situ keratomileusis with confocal microscopy. Ophthalmology 2001;108: 1744 –50. 28. Powers TP, Stewart WC, Stroman GA. Ultrastructural features of filtration blebs with different clinical appearances. Ophthalmic Surg Lasers 1996;27:790 – 4. 29. Addicks EM, Quigley HA, Green WR, Robin AL. Histologic characteristics of filtering blebs in glaucomatous eyes. Arch Ophthalmol 1983;101:795– 8. 30. Sacu S, Rainer G, Findl O, et al. Correlation between the early morphological appearance of filtering blebs and outcome of trabeculectomy with mitomycin C. J Glaucoma 2003;12: 430 –5. 31. Jampel HD, McGuigan LJB, Dunkelberger GR, et al. Cellular proliferation after experimental glaucoma filtration surgery. Arch Ophthalmol 1988;106:89 –94. 32. Chen CW. Enhanced intraocular pressure controlling effectiveness of trabeculectomy by local application of mitomycin C. Trans Asia-Pac Acad Ophthalmol 1983;9:172–7. 33. Shields MB, Scroggs MW, Sloop CM, Simmons RB. Clinical and histopathological observations concerning hypotony after trabeculectomy with adjunctive mitomycin C. Am J Ophthalmol 1993;116:673– 83.
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Figure 2. In vivo confocal microscopy images of (A) normal conjunctival and (B) corneal cells (400⫻400 m).
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Trabeculectomy has become the method of choice in surgical treatment of patients with glaucoma.1 This surgical technique is usually associated with an elevation of the conjunctiva overlying the scleral flap (i.e., the filtering bleb). The long-term success of filtering surgery is not only dependent on surgical technique; age, type of glaucoma,2 ethnic origin, prior failed filtering surgery or long-term use of topical medications before surgery3 are also critical factors. However, the development of a filtering bleb, determined by the postoperative wound healing process, is a major factor of efficiency and long-term success of surgical procedures. For these reasons, many authors have investigated Originally received: January 27, 2005. Accepted: May 29, 2005.
Manuscript no. 2005-88.
1
Department of Ophthalmology III, Quinze-Vingts National Ophthalmology Hospital, Paris, France.
2
INSERM U598, University of Paris 5, Paris, France. Department of Ophthalmology, Ambroise Paré Hospital, APHP, University of Versailles, Versailles, France. Supported by Quinze-Vingts National Ophthalmology Hospital, Paris, France. Correspondence to Christophe Baudouin, MD, PhD, Service d’Ophtalmologie III, C.H.N.O. des Quinze-Vingts, 28 rue de Charenton, Paris, 75012 France. E-mail: [email protected]. 3
© 2005 by the American Academy of Ophthalmology Published by Elsevier Inc.
morphologic criteria of these blebs to correlate clinical and functional aspects.4,5 However, in some cases, there is no correlation between bleb appearance or shape and intraocular pressure. The reasons for bleb failure, related to bleb scarring and histological changes within the bleb tissue, are not easy to study in clinical practice. In vivo confocal microscopy can provide details of ocular structures at the cellular level, and it has already been used in numerous studies of normal and pathologic corneas. Recent advances in the in vivo confocal microscopy technique have permitted visualization of peripheral ocular structures. However, in vivo confocal microscopy is a relatively new method for investigating conjunctiva.6 In a preliminary report, we have presented the technique allowing the description of blebs using in vivo confocal microscopy.7 The aim of the present study was to quantify and to compare, in a masked manner the in vivo confocal microscopy aspects of functioning and nonfunctioning blebs, using the new confocal microscope Heidelberg Retina Tomograph II/Rostock Cornea Module (Heidelberg Engineering GmbH, Heidelberg, Germany) to provide a better understanding of filtering mechanisms and the wound healing process after filtering surgery. ISSN 0161-6420/05/$–see front matter doi:10.1016/j.ophtha.2005.05.021
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Patients and Methods In this pilot study, we retrospectively evaluated 17 filtering blebs of 13 patients who had previously undergone trabeculectomy, and who were followed-up at the Quinze-Vingts National Ophthalmology Hospital, Paris, France. Six of the patients were female (46.1%) and 7 were male (53.9%), whose ages ranged from 21 to 79 (mean⫾standard deviation; 55.5⫾16.4 years). There were 10 right eyes (58.9%) and 7 left eyes (41.1%). Of the 17 eyes, 15 (88.2%) had primary open-angle glaucoma and 2 (11.8%) had juvenile open-angle glaucoma. In 5 cases (29.4%), mitomycin C (MMC) had been intraoperatively applied for 2 minutes using a small piece of surgical sponge soaked in 0.2 mg/ml MMC. After exposure, the conjunctival flap area was immediately irrigated with 250 ml balanced salt solution (BSS Alcon, Fort Worth, TX). The period between surgery and in vivo confocal microscopy evaluation ranged from 1 to 228 months (mean⫾standard deviation; 47.9⫾63 months). First, all patients had a slit-lamp examination and Goldmann applanation tonometry. This study was performed in compliance with French regulations on biomedical research, and all individual patients were informed of the aims of recording these data, and their consent was obtained. Patients were examined using a new in vivo confocal microscope (i.e., the Heidelberg Retina Tomograph (HRT) II/Rostock Cornea Module [Heidelberg Engineering GmbH, Heidelberg, Germany]). The HRT II is a confocal scanning laser ophthalmoscope that was initially developed for the analysis of the posterior pole of the eye, especially the optic nerve head, and it has become a standard for investigating glaucomatous changes of the optic disc.8,9 With the addition of the Rostock Cornea Module, the HRT II is converted to an in vivo confocal microscope available for investigating the ocular surface.10 Before the in vivo confocal microscopy examination, 1 drop of topical anesthetic (Novesine 0,4% [oxybuprocaïne 0.4 %] MSD-Chibret, Paris, France) and 1 drop of gel tear substitute (Lacrigel, carbomer 0,2%, Europhta, Monaco) are instilled into the lower conjunctival fornix. The patient is then seated at an examination table with the head into the headrest. The patient fixates with the contralateral eye at a small bright red light. The adjustment of the eye is performed by means of the live image and under control of a charged-coupled device color camera (640 ⫻ 480 pixels, RGB, 15 frames/s). The x-y position of the image and the section depth are controlled manually. The objective of the microscope is an immersion lens (Olympus, Hamburg, Germany), magnification ⫻60, covered by a polymethyl metacrylate cap. The laser source used in the HRT II/ Rostock Cornea Module is a diode laser with a wavelength of 670 nm. Images consist of 384 ⫻ 384 pixels covering an area of 400 m ⫻ 400 m, with transversal optical resolution of 2 m and longitudinal optical resolution of 4 m (Heidelberg Engineering). For all eyes, several confocal microscopic images of the superficial epithelium and of the subepithelial connective tissue of the conjunctiva located over the trabeculectomy site were taken. Each eye was examined for less than 5 minutes and no complications related to in vivo confocal microscopy evaluation were noted. Eyes were divided into 3 groups: (1) functioning blebs (6 eyes; 35.3%), (2) nonfunctioning blebs (6 eyes; 35.3%), and (3) functioning blebs after MMC application (5 eyes; 29.4%). In the nonfunctioning bleb group (group 2), 2 clinical appearances were analyzed: (1) flat blebs (5 blebs) and (2)encapsulated blebs (1 bleb). Surgical success of filtering surgery was defined by the intraoc-
ular pressure (IOP), according to previous studies.5,11,12 Functioning blebs were defined as IOP ⬍21 mmHg without antiglaucoma treatment, and nonfunctioning blebs were defined as IOP ⱖ21 mmHg and/or the necessity for an antiglaucoma medication. To ensure consistency, images were analyzed retrospectively by a single researcher (CB) who was masked toward clinical features and IOP control. In vivo confocal microscopy images were evaluated for conjunctival and corneal epithelium changes, presence and number of intraepithelial microcysts rated from 0 (none) to 3 (numerous) (Fig 1A), size of microcysts classified as ⬍100 m and ⬎100 m, density of subepithelial connective tissue rated from 0 (loosely) to 3 (dense) (Fig 1B), presence of blood vessels, or encapsulation of the bleb. Statistical values of number of microcysts and density of subepithelial connective tissue (mean⫾standard error of the mean (SEM) were calculated in each group and compared using the nonparametric Mann–Whitney U test. Probability values less than 0.05 were considered significant.
Results For group 1, the functioning blebs in vivo confocal microscopy showed several distinct characteristics. Conjunctival and corneal epithelial cells were perfectly seen and had normal appearance compared with normal eyes13 or patterns found in areas distant from the blebs (Fig 2 [available at http://aaojournal.org]). Between normal epithelial cells, numerous optically clear spaces filled with fluid were seen corresponding to microcysts as observed with the slit lamp (Fig 3A). These microcysts were particularly numerous in functioning blebs, with all the functioning blebs having a number of microcysts rated 2 or 3 (mean⫾SEM, 2.5⫾0.22; P ⫽ 0.0001, as compared with nonfunctioning blebs, mean⫾SEM, 0.667⫾0.21), and were predominantly found in the conjunctiva adjacent to the limbus. The size of the microcysts was between 10 and 150 m in 83.3% of the functioning blebs. Subepithelial connective tissue images showed a loosely arranged tissue, with all the functioning blebs having a density of connective tissue rated 0 or 1 (mean⫾SEM, 0.667⫾0.21; P ⫽ 0.001, as compared with nonfunctioning blebs, mean⫾SEM, 2.5⫾0.34). This tissue was widely spaced and contained clear spaces (Fig 3B). No blood vessel was clearly seen in the subepithelial connective tissue. Regarding group 2, that of the nonfunctioning blebs, conjunctival and corneal cells were again perfectly seen with normal appearance. Very few optical clear spaces corresponding to microcysts were observed between superficial conjunctival cells (mean⫾SEM, 0.667⫾0.21; P ⫽ 0.0001, as compared with functioning blebs, mean⫾SEM, 2.5⫾0.22), and all the nonfunctioning blebs had microcysts rated 0 or 1 (Fig 4A). Three (50%) of the nonfunctioning blebs showed few microcysts containing optically dense material (Fig 4B). Within the clinically encapsulated nonfunctioning bleb, dense fibrotic tissue evocative of encapsulation was observed (Fig 4C). Conversely, an encapsulation-like pattern was observed with in vivo confocal microscopy in 1 clinically flat nonfunctioning bleb. In an encapsulated bleb, microcysts were observed mostly in the peripheral area of the bleb. The subepithelial tissue of nonfunctioning blebs showed a dense, connective tissue with few or no clear spaces, with 83.3% of these blebs having a density that was rated 2 or 3 (mean⫾SEM, 2.5⫾0.34;, P ⫽ 0.001, as compared with functioning blebs, mean⫾SEM,
™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™3 Figure 1. In vivo confocal microscopy images of blebs (400 m⫻400 m). A, Rating of microcysts in the conjunctival epithelium: 0, 1, 2, or 3. B, Rating of density of subepithelial connective tissue: 0, 1, 2, or 3.
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Figure 3. In vivo confocal microscopy images of functioning blebs (400 m⫻400 m). A–D, Examples of numerous microcysts in the conjunctival epithelium. E–H, Examples of subepithelial connective tissue widely spaced, containing clear spaces.
0.667⫾0.21) (Fig 4D). Blood vessels were seen in the subepithelial connective tissue of 4 (66.7%) nonfunctioning blebs (Fig 4D). Regarding group 3, the functioning blebs with MMC, we observed numerous clear spaces corresponding to large confluent microcysts (Fig 5A) between morphologically normal conjunctival epithelial cells. Four of 5 of these blebs had a number of microcysts rated 2 or 3 (mean⫾SEM, 2.2⫾0.22; P ⫽ 0.001, as compared with nonfunctioning blebs, mean⫾SEM, 0.667⫾0.21). These microcysts had different sizes, ranging from 10 to 300 m. Some of these microcysts showed hyper-reflective microdots in the superficial epithelium layer (Fig 5A) in 3 blebs. The nature of these hyper-reflective dots remains unknown, but they might have been necrotic epithelial cells or inflammatory cells. The subepithelial tissue was characterized by loosely arranged connective tissue
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with numerous large clear spaces (Fig 5B), and 4 of 5 of these blebs had a density of subepithelial tissue rated 0 or 1 (mean⫾SEM, 0.8⫾0.37; P ⫽ 0.0084, as compared with nonfunctioning blebs, mean⫾SEM, 2.5⫾0.34). In one of these blebs, an encapsulation was observed with in vivo confocal microscopy. No blood vessel was observed in the subepithelial tissue of functioning blebs with MMC. These results and bleb confocal microscopy image characteristics are shown in Table 1.
Discussion The long-term success of trabeculectomy is mainly dependent on the development of a functioning bleb. The forma-
Labbé et al 䡠 In Vivo Confocal Microscopy and Filtering Blebs
Figure 4. In vivo confocal microscopy images of nonfunctioning blebs (400 m⫻400 m). A–C, Examples of blebs having none or few microcysts in the conjunctival epithelium. D, E, Examples of microcysts containing optically dense material. F, Encapsulation observed at the periphery of the bleb. G, H, Examples of dense subepithelial connective tissue. I, Blood vessels in the subepithelial tissue.
tion and the maintenance of this functioning bleb, with regard to wound healing and conjunctival scarring, are therefore of primary importance. For these reasons, many authors have presented classifications of these blebs to correlate the morphologic criteria observed biomicroscopically with the outcome of these blebs. Picht and Grehn11,12 classified the developing, filtering bleb, showing that favorable bleb development was characterized by microcysts of the conjunctiva, paucity of vessels, diffuse bleb, and moderate elevation of the bleb. In contrast they observed that unfavorable bleb development was characterized by increased vascularization, cork screw vessels, encapsulation
of the bleb and high-domed appearance. Because in some cases the appearance of the bleb is not correlated to IOP, and because the reason of failure is often unclear, some authors have looked for new in vivo evaluation techniques such as ultrasound biomicroscopy14 –16 to understand bleb failure mechanisms. However, ultrasound biomicroscopy evaluation or morphologic criteria observed biomicroscopically can only indirectly observe and cannot analyze histological changes explaining bleb failure. The principle of confocal microscocopy was first described by Minsky in 195717 The technological advances of the past 40 years, have led to the development of in vivo
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Figure 5. In vivo confocal microscopy images of functioning blebs with mitomycin C (MMC) (400 m⫻400 m). A, B, Examples of large, confluent, and numerous microcysts. C, D, Microcysts containing hyper-reflective microdots. E, F, Examples of loosely arranged connective tissue.
confocal microscopy for observation of the human cornea under normal6,18 or pathological conditions.19 –27 More recently, the conjunctival epithelium and stroma could also be observed at the cellular level using a new-generation in vivo confocal microscope.6 In a preliminary report, we described the first patterns of blebs after filtering surgery using this new technique of in vivo confocal microscopy.7 In the present study, we further analyzed these findings more precisely and compared the different types of blebs observed after trabeculectomy. The purpose of this study was to correlate the images obtained with in vivo confocal microscopy, with regard to the number of microcysts, the subepithelial tissue density, and the presence of encapsulation or vessels, with the morphological and functional aspects of these blebs. Functioning blebs (group 1) showed a normal conjunctival epithelium with numerous microcysts and a subepithelial tissue arranged loosely and hypo-reflective, with a high number of optically clear spaces. In contrast, nonfunctioning blebs (group 2) showed none or very few microcysts, and in this case, the subepithelial tissue was hyper-reflective, with dense collagenous connective tissue and blood vessels. For Powers et al,28 who studied functioning blebs using light and electron microscopy, the subepithelial connective tissue was loosely arranged with widely spaced collagen. These authors also described clear spaces in the superficial substantia. In a study of blebs using light and electron
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microscopy, Addicks et al29 showed that nonfunctioning blebs had dense collagenous connective tissue. For these authors, functioning blebs had looser subepithelial connective tissue with histologically clear spaces corresponding to mycrocysts. Our results are in good consistency with ex vivo histological analyses of functioning and nonfunctioning blebs previously described. The presence and number of these microcysts are assumed to be a positive predictive factor for functioning blebs, and interestingly these microcysts were observed in this study as early as the first month after surgery. The morphologic analysis of functioning blebs frequently showed these microcysts.5,11,30 The in vivo confocal microscopic examination of functioning blebs seems to add strength to the argument that these microcysts are channels for the passage of aqueous humor.30 It has been well known for some time that the subepithelial connective tissue of the blebs has a major influence on IOP control. Fibrotic scarring in the subconjunctival space and excessive extracellular matrix production has been previously observed in nonfunctioning blebs.29 –31 In this study, in vivo confocal microscopy images of functioning blebs showing loosely arranged connective tissue and nonfunctioning blebs showing dense subepithelial tissue were well consistent with previous histological studies.28,29 Mitomycin C is applied during filtering surgery to reduce the risk of bleb failure.32 By inhibiting cell proliferation, MMC prevents an excessive healing response
Labbé et al 䡠 In Vivo Confocal Microscopy and Filtering Blebs Table 1. Individual Data and In Vivo Confocal Microscopy Characteristics In Vivo Confocal Microscopy Evaluation Microcysts No.
Eye
Follow-up Period (mos)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
R R L R R R R L L L L R R R L R L
15 228 60 5 96 27 30 28 6 120 144 43 6 2 2 1 1
Connective tissue
Classification of Blebs
Quantity (0 –3)
Size*
Characteristics of Microcysts
Density (0 –3)
Encapsulation
Vessels
F F F F F F Non F, En Non F Non F Non F Non F Non F F with MMC F with MMC F with MMC F with MMC F with MMC
3 3 2 2 2 3 1 1 1 0 0 1 3 2 3 1 2
⬍ ⬍ ⬍ ⬍ ⬍ ⬎ ⬍ ⬍ ⬍ — — ⬍ ⬎ ⬎ ⬎ ⬍ ⬍
E E E E E E E D D — — D E, HMD E, HMD E, HMD E E
1 1 1 1 0 0 3 1 2 3 3 3 0 1 0 2 1
N N N N N N Y N N N Y N N N N Y N
N N N N N N N N Y Y Y Y N N N N N
*Size of microcysts: ⬍100 m; ⬎100 m. D ⫽ dense; E ⫽ empty; F ⫽ functioning bleb; F with MMC ⫽ functioning bleb with mitomycin C; HMD ⫽ Hyper-reflective microdots; L ⫽ left; N ⫽ no; Non F ⫽ nonfunctioning bleb; Non F, En ⫽ nonfunctioning clinically encapsulated bleb, R ⫽ right; Y ⫽ yes.
and scarring, and thus enhances the success of the procedure. Shields et al33 have used light and electron microscopy to analyze an excised bleb after trabeculectomy with MMC; they found numerous microcysts within the epithelium and a loosely arranged connective tissue within the subepithelium. Necrotic nuclei and cytoplasmic vacuoles were also observed in the epithelium of these blebs using electron microscopy.29 The hyper-reflective microdots observed with in vivo confocal microscopy between and within microcysts would likely correspond to these necrotic nuclei. In our study, in vivo confocal microscopy images of functioning blebs with MMC well agreed with previous ex vivo analyses. In vivo confocal microscopy evaluation of blebs after filtering surgery thus allowed us to study, at a cellular level, the conjunctival wall of these blebs. By providing in vivo high-resolution images of these structures, we obtained results similar to ex vivo microscopic analyses. However, we observed a nonfunctioning bleb, according to clinical criteria, with a loosely arranged connective tissue, and one functioning bleb with MMC that had few microcysts. These few cases probably demonstrate the limits of this evaluation. Only a limited part of the conjunctiva covering the bleb is examined at a time, and one cannot make conclusions from such focal examination. Nevertheless, in vivo confocal microscopy evaluation is now providing new insight into the analysis of filtering blebs. This new method is rapid and noninvasive and it allows clinicians and researchers to visualize in vivo functioning or nonfunctioning blebs at the cellular level. Using this technique we can observe directly the histological processes that correlate with filtration or failure. By studying the wound healing mechanisms after filter-
ing surgery in vivo, this technique will permit a better understanding of filtration failure. Clinicians, with images at a cellular level, would be able to predict the outcome of these blebs and eventually provide specific treatments to enhance success rates of their surgical procedures.
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22. Pfister DR, Cameron JD, Krachmer JH, Holland EJ. Confocal microscopy findings of Acanthamoeba keratitis. Am J Ophthalmol 1996;121:119 –28. 23. Rosenberg ME, Tervo TM, Muller lJ, et al. In vivo confocal microscopy after herpes keratitis. Cornea 2002;21:265–9. 24. Ciancaglini M, Carpineto P, Zuppardi E, et al. In vivo confocal microscopy of patients with amiodarone-induced keratopathy. Cornea 2001;20:368 –73. 25. Mustonen RK, McDonald MB, Srivannaboon S, et al. In vivo confocal microscopy of Fuch’s endothelial dystrophy. Cornea 1998;17:493–503. 26. Werner LP, Werner L, Dighiero P, et al. Confocal microscopy in Bowman and stromal corneal dystrophies. Ophthalmology 1999;106:1697–704. 27. Pisella PJ, Auzerie O, Bokobza Y, et al. Evaluation of corneal stromal changes in vivo after laser in situ keratomileusis with confocal microscopy. Ophthalmology 2001;108: 1744 –50. 28. Powers TP, Stewart WC, Stroman GA. Ultrastructural features of filtration blebs with different clinical appearances. Ophthalmic Surg Lasers 1996;27:790 – 4. 29. Addicks EM, Quigley HA, Green WR, Robin AL. Histologic characteristics of filtering blebs in glaucomatous eyes. Arch Ophthalmol 1983;101:795– 8. 30. Sacu S, Rainer G, Findl O, et al. Correlation between the early morphological appearance of filtering blebs and outcome of trabeculectomy with mitomycin C. J Glaucoma 2003;12: 430 –5. 31. Jampel HD, McGuigan LJB, Dunkelberger GR, et al. Cellular proliferation after experimental glaucoma filtration surgery. Arch Ophthalmol 1988;106:89 –94. 32. Chen CW. Enhanced intraocular pressure controlling effectiveness of trabeculectomy by local application of mitomycin C. Trans Asia-Pac Acad Ophthalmol 1983;9:172–7. 33. Shields MB, Scroggs MW, Sloop CM, Simmons RB. Clinical and histopathological observations concerning hypotony after trabeculectomy with adjunctive mitomycin C. Am J Ophthalmol 1993;116:673– 83.
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Figure 2. In vivo confocal microscopy images of (A) normal conjunctival and (B) corneal cells (400⫻400 m).
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