Dear Editor

We would like to thank Dr Rodrigues and colleagues for bringing up this interesting point of what exactly trypan blue (TB) stains.

In our study, immunohistochemistry was performed to determine the nature of cells involved in the epiretinal membranes (ERM) - not to determine the presence or absence of the ERM. Presence or absence of ERM was determined by examining routinely stained sections (H&E, PAS) for cytoplasm/nuclei of epiretinal cell elements. All four of the macular hole internal limiting membrane (ILM) specimens were examined in this way.[1] Furthermore TB (in low concentrations) stains the anterior lens capsule.[2] Since this capsule lacks glia, we do not believe that the evidence supports the contention of the correspondents that the staining of our ILM specimens is due to undetected "glial cell elements of the highly cellular ERM" rather than ILM.

Clinically two features are observed with the use of TB. Firstly, the whole posterior pole that comes into contact with TB is stained a faint blue in all cases. The staining pattern is diffuse and not patchy, suggesting TB straining is indiscriminate of ERM or ILM. Secondly, in cases of macular pucker, the TB stained ERM can be removed separately, leaving intact ILM behind, which can be further stained and removed. In cases of macular hole where a clinical ERM is not present, it appears that only the ILM is stained and peeled. We have harvested these membranes and confirmed that the membranes only consist of ILM and without a secondary ERM.

There is no doubt that TB stains both ERM and ILM. We however, have no knowledge as to what the structural elements of these membrane that the dye is attached to. We concede that staining of ILM with TB can be variable and sometimes rather faint. Since our publication, Dr. Perrier and Dr. Sebag have also reported their experience with TB in staining ILM and ERM.[3,4] Although histological findings were not given in these studies, clinically the authors found the dye to be useful in both types of membranes. Given the many concerns regarding the use of indocyanine green,[5] we believe it is a positive development that an alternative clinically useful dye is available.

References

(1) Li K, Wong D, Hiscott, et al. Trypan blue staining of internal limiting membrane and epiretinal membrane during vitrectomy: visual results and histopathological findings. Br J Ophthalmol 2003;87:216-19.

(2) Melles GR, de Waard PW, Pameyer JH, et al. Typan blue capsule staining to visualize the capsulorhexis in cataract surgery. J Cataract Refract Surg 1999;25:7–9.

(3) Perrier M, Sebag M. Trypan blue-assisted peeling of the internal limiting membrane during macular hole surgery. Am J Ophthalmol 2003;135(6): 903-05.

(4) Perrier M, Sebag M. Epiretinal membrane surgery assited by typan blue. Am J Ophthalmol 2003;135(6):909-11.

(5) Gandorfer A, Haritoglou C, Gass CA, et al. Indocyanine green- assisted peeling of the internal limiting membrane may cause retinal damage. Am J Ophthalmol 2001;132:431–3.

Dear Editor

We read with great interest the manuscript by Li et al. about staining of the internal limiting membrane (ILM) and epiretinal membrane (ERM) with trypan blue (TB).[1] We would like to comment one aspect of this manuscript, when the authors claimed that a good staining of both the ILM and the ERM was achieved with TB. We disagree that ILM is stained by TB, and propose that TB only stains the ERM, but not the ILM.

The authors affirm "ILM staining" with TB as they observed histologically the presence of ILM in four eyes with macular holes stage III and IV. In one of those eyes, immunohistochemistry was performed, and an epiretinal membrane was observed. The other 3 cases, immunohistochemistry examination was not made because of insufficient tissue. Most of the stage III and IV macular holes are known to be associated with an epiretinal membrane,[2] and probably an ERM would be seen in addition to the ILM in those 3 cases if immunohistochemistry for glial elements were performed. Therefore, we believe that TB stained the ERM associated with the macular holes, but not the ILM. In their study, staining with TB of 7 patients with idiopathic epiretinal membrane was successfully performed. ERM of proliferative vitreoretinopathy is also reported to be well stained by TB.[3] We speculate that TB has binding affinity to some of the glial cell elements of the highly cellular ERMs, either those associated with macular holes or not.

Indocyanine green (ICG) is another new dye for intraocular staining, that gained wide acceptance among retina surgeons in the last few years.[4] In contrast to the cellular affinity of TB, ICG stains well the acellular ILM, because of the fast binding of ICG to collagen proteins of the ILM. ERM tends to stain negatively by ICG, because the hydrophilic ICG does not penetrate cell membranes easily.[5]

TB staining seems to be a good alternative to ICG staining in the surgical management of macular diseases. Further studies are warranted about the intraocular kinetics of that dye.

References

(1) Li K, Wong D, Hiscott P, et al. Trypan blue staining of internal limiting membrane and epiretinal membrane during vitrectomy: visual results and histopathological findings. Br J Ophthalmol 2003;87:216-19.

(2) Tornambe P, Augustin AJ: Macular holes. Review of the current status of knowledge of pathogenesis and treatment methods. Ophthalmologe 2002;99:601-08.

(3) Feron EJ, Veckeneer M, Parys-Van Ginderdeuren R, et al. Trypan blue staining of epiretinal membranes in proliferative vitreoretinopathy. Arch Ophthalmol2002;120:141-4.

(4) Schmidt JC, Meyer CH, Rodrigues EB, et al. Staining of internal limiting membrane in vitreomacular surgery: A Simplified technique. Retina 2003 In Press.

(5) Forster RE, Petersen MR, Da Mata AP, et al. Negative indocyanine green staining of epiretinal membranes. Retina 2002;22:106-7.

Dear Editor

The authors wish to thank Gandorfer and colleagues for their interest in our paper [1] and for the kind comments and encouragement with regard to our work. Certainly, these correspondents are compiling evidence concerning the effect of indocyanine green (ICG) on the retina in both their published and unpublished studies.[2,3]

In our report, we restricted our comments regarding retinal damage and dye usage to the specimens wherein an epiretinal membrane (ERM) was present.[1] Evidence of retinal damage was observed in four of these five specimens, mostly in the form of neural and glial elements adherent to the retinal side of the inner limiting membrane (ILM). The apparent lack of such elements in some ERM specimens may reflect partial separation of the ILM due to traction from the membrane prior to surgery. [4] Nevertheless, in one of our specimens a substantial fragment of neuroretina was also present.[1] We have, indeed, long considered such fragments as potential confounding factors in immunohistochemical studies of surgically-excised ERMs.[5,6] Since they are present in ERMs removed without the use of any dye at all, we cannot blame their presence on these surgical aids. Perhaps these fragments are avulsed as a result of enhanced adhesion between the ERM and retina via glial anchorage sites running through dehiscences in the ILM.[5] It is clear that our investigation does not exclude an effect of ICG on the retina [2,3] and we wholeheartedly agree with Gandorfer and coworkers that agents such as trypan blue warrant further evaluation as aids to ERM and ILM peeling.

References

(1) Li K, Wong D, Hiscott P, Stanga P, Groenewald C, McGalliard J. Trypan blue staining of internal limiting membrane and epiretinal membrane during vitrectomy: visual results and histopathological findings. Br J Ophthalmol 2003;87:216-9.

(2) Gandorfer A, Haritoglou C, Gass CA, Ulbig MW, Kampik A. Indocyanine green-assisted peeling of the internal limiting membrane may cause retinal damage. Am J Ophthalmol 2001;132:431-3.

(3) Gandorfer A, Haritoglou C, Gandorfer A, Kampik A. Retinal damage from indocyanine green in experimental macular surgery. Invest Ophthalmol Vis Sci 2003;44:316-23.

(4) Michels RG. A clinical and histopathologic study of epiretinal membranes affecting the macula and removed by vitreous surgery. Trans Am Ophthalmol Soc 1982;80:580-656.

(5) Hiscott PS, Grierson I, Trombetta C, Rahi AHS, Marshall J, McLeod D. Retinal and epiretinal glia an immunohistochemical study. Br J Ophthalmol 1984;68:698 707.

(6) Morino I, Hiscott P, McKechnie N, Grierson, I. Variation in epiretinal membrane components with clinical duration of the proliferative tissue. Br J Ophthalmol 1990;74:393 9.

Dear Editor

We read with interest the article by Dr Li and colleagues about trypan blue staining of the vitreomacular interface during vitrectomy [1]

We congratulate the authors on their work. In particular, we appreciate their critical approach of testing trypan blue for staining of the internal limiting membrane (ILM) and epiretinal membrane (ERM) as well as their comments on potential untowards effects of indocyanine green (ICG) in macular surgery. We would like to comment on two remarks concerning the ultrastructural findings on the retinal side of the ILM following ILM- removal with and without the use of ICG.

We agree with Dr Li and collegues that fragments of glial cells are commonly found in ILM-specimens. Ultrastructurally, they appear as tiny fragments of Müller cell membranes adherent to and enclosed within the undulations of the retinal side of the ILM. These glial structures had been described in detail by Eckhardt and collegues, and are in accordance with previous work of our group, in an investigation of the ultrastructure of the vitreomacular interface of 93 specimens in 91 consecutive patients with macular holes, epiretinal membranes, diffuse diabetic macular edema, and vitreomacular traction syndrome without the use of indocyanine green or other dyes (unpublished data).[2] We also agree with the authors, that the surgical technique and the underlying disease may influence the amount of glial structures adherent to the retinal side of the ILM, as these structures are predominantly found within undulations and folds of the ILM.[3,4] However, we would like to emphasize the effect of ICG in this context. Firstly, there are obvious differences between ILM specimens removed with and without the use of ICG not only in terms of quantity of glial structures but in terms of quality. A continuous layer of cell membranes, undetermined cellular debris, and entire footplates of Müller cells were commonly observed following ICG-assisted peeling of the ILM, whereas such structures had never been found in the series of 93 unstained specimens described above.[5] Secondly, all stained and unstained specimens having been investigated by electron microscopy were removed by one experienced surgeon (A.K.). Beside the use of ICG, there was no change of the surgical technique. Moreover, retinal elements as described above were not found before the introduction of the dye at our institution in September 2000, nor after having stopped ICG-staining in April 2001. Thirdly, in an experimental setting in human donor eyes published recently, retinal structures adherent to the undulating side of the ILM as described above could be found following the application of ICG to the macula only.5 No attempt of peeling or any other mechanical approach to the vitreomacular interface was made in these eyes. However, the ILM was detached from the macula. Retinal elements were adherent to the retinal side of the ILM showing an identical morphology like those obtained during vitrectomy with ICG-assisted ILM-removal.[6] Therefore, in our experience, there is increasing evidence that at least some commonly used preparations of ICG may affect the ultrastructure of the inner retina, and are primarily responsible for obvious differences in the ultrastructure of the surgically removed ILM. ILM-removal by itself results in removal of tiny fragments of Müller cell membranes. Their morphological and functional implications to the macula remain unknown. Finally, we would like to encourage the authors to follow-on their promising approach of staining the ILM and ERM with trypan blue. In our institution, single specimens which had been stained and peeled using trypan blue revealed no evidence of retinal damage (submitted data).

References

(1) K Li, D Wong, P Hiscott, P Stanga, C Groenewald, and J McGalliard. Trypan blue staining of internal limiting membrane and epiretinal membrane during vitrectomy: visual results and histopathological findings. Br J Ophthalmol 2003;87:216-219.

(2) Eckardt C, Eckardt U, Groos S, Luciano L, Reale E. [Removal of the internal limiting membrane in macular holes. Clinical and morphological findings]. Ophthalmologe 1997;94:545-51.

(3) Haritoglou C, Gandorfer A, Gass CA, Kampik A. Ultrastructure of epiretinal tissue removed during indocyanine green assisted peeling in macular pucker surgery [ARVO Abstract No 3516]. Invest Ophthalmol Vis Sci 2002.

(4) Haritoglou C, Gandorfer A, Gass CA, Schaumberger M, Ulbig M, Kampik A. Indocyanine green-assisted peeling of the internal limiting membrane in macular hole surgery affects visual outcome: a clinicopathologic correlation. Am J Ophthalmol 2002;134:836-41.

(5) Gandorfer A, Haritoglou C, Gass CA, Ulbig MW, Kampik A. Indocyanine green-assisted peeling of the internal limiting membrane may cause retinal damage. Am J Ophthalmol 2001;132:431-3.

(6) Gandorfer A, Haritoglou C, Gandorfer A, Kampik A. Retinal damage from indocyanine green in experimental macular surgery. Invest Ophthalmol Vis Sci 2003;44:316-23.