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Abstract
Aims To evaluate the intraoperative applicability and safety of a mixture of brilliant blue G and sodium hyaluronate (visco-BBG) for staining the inner limiting membrane (ILM).
Methods A retrospective consecutive case series. Seventy-four eyes that had undergone ILM peeling were studied. During vitrectomy, ILM peeling with visco-BBG (visco-BBG group) was performed on 40 eyes; 12 with a macular hole (MH), 26 with an epimacular membrane (ERM) and 2 with a retinal detachment due to a MH (MHRD). ILM peeling with BBG dissolved in balanced salt solution (BSS-BBG group) was performed on 34 eyes; 9 with a MH, 23 with an ERM and 2 with a MHRD. The main outcome measures were the distribution of the dye within the vitreous cavity and the retinal sensitivity in the MH patients of the two groups by microperimetry.
Results The visco-BBG was injected over the retina where the ILM was intended to be peeled, and it stained the ILM in all cases. It did not disperse throughout the vitreous cavity or into the subretinal space. The BSS-BBG dispersed throughout the vitreous cavity, and its distribution was difficult to control. The two solutions did not stain the epiretinal membranes or any residual posterior hyaloid membrane. The difference in the retinal sensitivity between the two patients with MH of two groups was not significant. No complications were found in the visco-BBG group, although an accidental retinal perforation was found in one eye of the BSS-BBG group. Transmission electron microscopy confirmed that the membrane peeled was the ILM.
Conclusions Visco-BBG can be a useful method to assist macular surgery and can overcome some of the disadvantages of conventional BBG solutions dissolved in BSS.
- Imaging
- Pharmacology
- Retina
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Introduction
Brilliant blue G (BBG) is used during intraocular surgery to make the internal limiting membrane (ILM) more visible.1 To achieve good staining of the ILM, the BBG solution must come in contact with the ILM, however when the conventional BBG solution is injected into the vitreous, it disperses throughout the vitreous cavity (see online supplementary video 1). When the adjuvant is jetted strongly against the ILM, a retinal perforation can occasionally occur (see online supplementary video 2).2 ,3
We have developed a new method to overcome the dispersion problem by mixing BBG with sodium hyaluronate, a viscoelastic material, and we have called this mixture visco-BBG. The purpose of this study was to determine the intraoperative applicability and safety of visco-BBG when it is used to stain the ILM.
Materials and methods
We reviewed the medical charts of 74 consecutive eyes that had undergone ILM peeling with visco-BBG or BBG dissolved in only balanced salt solution (BSS-BBG). All surgeries were performed at the Yokohama City University Hospital.
Par plana vitrectomy (PPV) combined with ILM peeling with visco-BBG was used on 40 eyes (visco-BBG group) from April 2010 to March 2011, and with BSS-BBG on 34 eyes (BSS-BBG group) from April 2009 to September 2010. The visco-BBG group consisted of 12 eyes with a macular hole (MH), 26 with an epimacular membrane (ERM) and 2 with a retinal detachment due to a MH (MHRD). The BSS-BBG group consisted of 9 eyes with a MH, 23 with an ERM and 2 with a MHRD.
This study was a sequential, non-randomised study, and the selection of which BBG solution was used was not performed randomly. In the earlier operations, BSS-BBG was mainly used and in the later operations visco-BBG was mainly used. All patients were followed for 6 months postoperatively.
The procedures used were approved by the Institutional Review Board of the Yokohama City University Hospital, and they conformed to the tenets of the Declaration of Helsinki. The possible risks of this treatment were explained to the patients before the surgery, and written informed consent was obtained from all patients.
Visco-BBG
BBG 250 (BBG; Sigma–Aldrich, St Louis, Missouri, USA) was dissolved in BSS (BSS plus; Alcon, Osaka, Japan) to a concentration of 1 mg/ml and sterilised in an autoclave. The solution was then further sterilised by passing it through a 0.22 µm syringe filter in the operating room.
To make the visco-BBG solution, 0.6 ml of sodium hyaluronate (OPEGAN 0.6; Santen Pharmaceutical, Osaka, Japan, relative density (specific gravity), 1.0107 g/ml) was added to 0.2 ml of the BBG solution. The final BBG concentration was 0.25 mg/ml, and the final relative density of the solution was 1.0080 g/ml.
BBG dissolved in only BSS-BBG
BBG 250 was dissolved in BSS to a concentration of 0.25 mg/ml according to the method of Enaida et al1 and sterilised in an autoclave. The solution was then further sterilised by passing it through a 0.22 µm syringe filter in the operating room.
Surgical technique
The surgery consisted of standard three-port PPV with triamcinolone acetonide, which made the vitreous body more visible.4 After the vitrectomy, the ERM was removed (figure 1). Next, about 0.3 ml of the visco-BBG solution or about 0.5 ml of the BSS-BBG solution was injected into the fluid-filled vitreous cavity over the macular area and allowed to stay for about 30 s with the infusion temporary stopped. Then, it was sucked out with the vitreous cutter. The ILM was then clearly visible. If remnants of the posterior hyaloid membrane and/or an ERM were present, they were peeled along with the stained ILM. The surgery was completed by a gas tamponade in patients with MH and MHRD.
Fundus photograph of an eye with an epiretinal membrane (ERM). The ERM was peeled by the forceps before the inner limiting membrane peeling stained with a mixture of brilliant blue G and sodium hyaluronate (visco-BBG). The white particles on the retina are the triamcinolone acetonide crystals which were used to make the vitreous more visible. The arrow points to the peeled ERM.
Retinal sensitivity
Fundus-related microperimetry (MP) is a method to measure the functional retinal sensitivity. The MP-1 microperimeter (Nidek Technologies, Padova, Italy) measures the retinal sensitivity at several points in the patient's fundus and maps out the sensitivity. An infrared camera establishes and tracks the patient's fixation, and the resulting visual field is registered onto the corresponding fundus photograph. The central 20° was tested in patients with MH in the visco-BBG and BSS-BBG groups before and after surgery. Goldmann III stimuli and a 4–2 staircase strategy were used with 76 stimulus locations on a circular test grid. The central 20° was tested in patients with MH with dilated pupils of the two groups before and after surgery. We selected patients with MH in the visco-BBG and BSS-BGG groups because they have a normal retina except for the MH areas and also because their preoperative demographics were not significantly different.
Outcome measures
The MP-1 determination of the retinal sensitivities and optical coherence tomography (OCT) were performed before and 6 months postoperatively.
Histological examinations
All of the ILM specimens peeled from May 2010 to July 2010 in the visco-BBG group were sent for transmission electron microscopy (TEM) to verify that the membrane removed was the ILM. The specimens were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) and post-fixed in veronal acetate buffer osmium tetroxide (2%). The specimens were then dehydrated in an ethanol series and embedded in Epon 812 (TAAB Laboratories, Berkshire, England). Ultrathin sections were cut from two randomly selected MH and ERM blocks and they were doubly stained with uranyl acetate and lead nitrate. The sections were examined with a JEM-1200EX TEM (JEOL, Tokyo, Japan) operated at 80 kV.
Results
Comparison of distribution of BBG solution in vitreous cavity
In all cases, the visco-BBG was dropped onto the retina where the ILM was intended to be peeled, and it did not diffuse to other parts of the retina and into the subretinal space (see online supplementary video 3). The ILM in the area intended for peeling was selectively stained bright blue by the visco-BBG and safely peeled. Any remnants of the posterior hyaloid membrane were removed along with the stained ILM after the ERM was peeled (figure 2). After peeling the ILM, a difference in the colour of the retina in the area where the ILM was intact from where the ILM was peeled was clearly visible (figure 3). The visco-BBG could be easily sucked out with a vitreous cutter.
Fundus photograph of an eye with an epiretinal membrane (ERM). After the ERM was removed, the inner limiting membrane (ILM) was selectively stained a bright blue by a mixture of brilliant blue G and sodium hyaluronate (visco-BBG). It was safely peeled with the unstained residual posterior hyaloid membrane. The white particles on the retina are triamcinolone acetonide crystals which were used to make the vitreous more visible. The arrow points to residual posterior hyaloid membrane. The arrowhead points to the border between the area from which the ILM had been removed and the ILM that was intact.
Fundus photograph of a case of macular hole retinal detachment. After the inner limiting membrane (ILM) was removed, the difference in the appearance of the retinal surface between the area from which the ILM had been removed and the surrounding area can be clearly seen (arrowheads). The white particles on the retina are triamcinolone acetonide crystals which were used to make the vitreous more visible.
The BSS-BBG dispersed throughout the vitreous cavity, and it was difficult to control the movement of the BBG solution (see online supplementary video 1). To stain the ILM, we needed to spray the BSS-BBG on the retinal surface. The ILM in the area intended for peeling was not selectively stained a bright blue by the BSS-BBG. The areas where a residual ERM or posterior hyaloid membrane was present did not stain at this concentration of visco-BBG and BSS-BBG.
Retinal sensitivity
The mean preoperative retinal sensitivity in the central 20° was 903.6±374.8 dB in the BSS-BBG group and 844.5±225.2 dB in the visco-BBG group (p=0.66, unpaired t test). The mean postoperative retinal sensitivity in the central 20° was 923.6±352.8 dB in the BSS-BBG group and 911.3±282.8 dB in the visco-BBG group at 6 months (p=0.53, unpaired t test). The difference between the preoperative and postoperative retinal sensitivity in the central 20° was 20.0±273.5 dB in the BSS-BBG group and 66.8±403.6 dB in the visco-BBG group was not significant (p=0.38, unpaired t test).
OCT and ophthalmic examinations
OCT examination showed that all of the MHs were closed, and recurrences of an MH were not detected by ophthalmic examinations including slit-lamp microscopy, ophthalmoscopy and OCT examination for 6 months. Similarly, the MHRDs were reattached and the ERMs were removed, and no recurrences were detected during the follow-up period.
Complications
No ocular hypertension or undue inflammation, that is, endophthalmitis, attributable to residual visco-BBG was evident postoperatively. There were no alterations of the retinal pigment epithelium due to dye toxicity in all cases. An accidental retinal perforation occurred in an ERM case of the BSS-BBG group.2
Transmission electron microscopy
TEM confirmed that the membrane collected during the surgery was the ILM in all processed specimens (figure 4).
Transmission electron micrograph of the removed tissue showing characteristics of the inner limiting membrane (ILM). The retinal side of the tissue has an irregular surface (R), and the vitreous side is smooth (V). This confirmed the presence of the ILM. Scale bars represent 5 μm.
Discussion
Mixing the BBG with a dextrose solution or with deuterium oxide5 can create a solution of high specific gravity. But the physicochemical properties of visco-BBG are not only high specific gravity but also high elasticity, viscosity and surface tension. Moreover, the visco-BBG had the same optical properties as the vitreous and is not toxic or allergenic.6 Thus, the visco-BBG can be ‘poured’ onto the retina rather than streaming it against the retina due to its heavy specific gravity. In addition, it will not diffuse throughout the vitreous cavity and stain the selective area of the ILM which is intended to be peeled because of its cohesive property and higher surface tension. In addition, the visco-BBG will not disperse into the subretinal space due to its high surface tension. Therefore, it is a safer and better intraoperative adjuvant than the usual BSS-BBG for macula surgery.
We had the impression that the staining effects of visco-BBG are equal or better than BSS-BGG. It seems reasonable to assume that the visco-BBG will remain in the retinal surface (see online supplementary video 4) due to its high surface tension and high specific gravity, resulting in a better visualisation of the ILM.
In summary, our findings show that visco-BBG can be managed in a very controlled way over the intended region without spraying. In addition, it will not disperse and will remain in the intended region. Thus, we recommend visco-BBG be used for ILM peeling due to its safety and ease of intraoperative use.
Acknowledgments
The authors thank Professor Duco Hamasaki, Bascom Palmer Eye Institute, University of Miami, School of Medicine, Miami, Florida for his critical discussion and final manuscript revision.
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Files in this Data Supplement:
- Data supplement 1 - Online video 1
- Data supplement 2 - Online video 2
- Data supplement 3 - Online video 3
- Data supplement 4 - Online video 4
Footnotes
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Contributors RU: substantial contributions to conception and design, acquisition of data, analysis and interpretation of data; drafting the article and revising it critically for important intellectual content; and final approval of the version to be published. HN-S: substantial contributions to conception and design, acquisition of data; drafting the article and revising it critically for important intellectual content; and final approval of the version to be published. AM, NI: acquisition of data; drafting the article and revising it critically for important intellectual content; and final approval of the version to be published. FY: analysis and interpretation of data; drafting the article and revising it critically for important intellectual content; and final approval of the version to be published. NM: substantial contributions to conception and design; drafting the article and revising it critically for important intellectual content; and final approval of the version to be published.
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Commercial and proprietary interest None of the authors or their family members has any proprietary or financial interest in the instruments used in this study. This study has not been published or presented at any meeting.
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Funding None.
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Competing interests None.
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Patient consent Obtained.
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Ethics approval The procedures used were approved by the Institutional Review Board of the Yokohama City University Hospital and they conformed to the tenets of the Declaration of Helsinki.
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Provenance and peer review Not commissioned; externally peer reviewed.
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Data sharing statement All authors have full control of all primary data and they agree to allow for BJO to review their data upon request.