Aims To evaluate, using anterior segment optical coherence tomography (AS-OCT) and in vivo confocal microscopy (IVCM), the uveo-scleral aqueous humour (AH) outflow pathways after ultrasonic circular cyclocoagulation (UCCC).
Methods Forty-four patients with refractory glaucoma underwent 4 or 6 s UCCC (group 1, 24 eyes; group 2, 20 eyes). UCCC was successful when the preoperative intraocular pressure (IOP) reduced by one-third. AS-OCT and IVCM were performed at baseline and at month 1 to evaluate the sclera and conjunctiva. The main outcomes were mean intra-scleral hyporeflective spaces area (MIHSA: mm2) at AS-OCT, mean density and area of conjunctival microcysts (MMD: cysts/mm2; MMA: µm2) at IVCM. The relations between MIHSA, MMA and MMD with IOP were analysed.
Results Mean baseline IOP was 26.9±2.8 mm Hg in group 1 and 27.5±4.0 in group 2. Intra-scleral hyporeflective spaces and microcysts were observed in both groups, without significant differences in MIHSA, MMA and MMD. At month 1, UCCC was successful in 63.6% of patients (41.6% in group 1, 80% in group 2), and IOP reduced to 18.8±3.2 (30.1%) and 17.1±2.7 mm Hg (38.7%), respectively (p<0.001). MIHSA showed a twofold and threefold increase in group 1 and 2 (p<0.05), with a significant difference between groups (p<0.05). MMA and MMD increased in both groups (p<0.05), with values higher in group 2 (p<0.05). Significant relations were found between MIHSA and IOP in both groups (p<0.01).
Conclusions UCCC induced anatomical modifications of sclera and conjunctiva, which suggested that the trans-scleral AH outflow enhancement is one of the possible mechanisms exploited by ultrasounds to reduce IOP.
- Aqueous humour
- Sclera and Episclera
- Treatment Surgery
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In glaucoma refractory to conventional treatments the ciliary body destruction is still considered as a last resort treatment to reduce the intraocular pressure (IOP). Several procedures have been proposed over the years,1–5 with the trans-scleral (TS) photocoagulation remaining the most used approach.3
In the last decades of the twentieth century, high-intensity focused ultrasound (HIFU) was also proposed as a cyclo-destructive procedure (Sonocare, Inc., Ridgewood, New Jersey, USA).6–12 The major advantage of using high-operating frequency ultrasounds is to focus on a well defined volume, at the desired depth, thus limiting damage to neighbouring tissues. Despite the good IOP lowering efficacy,11 ,12 the procedure was abandoned because of its excessive complexity.
To overcome these limitations, a modified HIFU technique (ultrasonic circular cyclocoagulation (UCCC)) was recently proposed.13–18 The procedure appears safe, with an IOP reduction from 26% to 36% at 12 months.14 ,15 ,18
The aqueous humour (AH) inflow reduction following the thermic necrosis of ciliary epithelium seems to play the main role in the final IOP lowering.16 However, an increase of the suprachoroidal and TS AH outflow has also been hypothesised.13–17 The aim of our study was to investigate the modifications of the uveo-scleral outflow pathway after UCCC in refractory glaucoma by using anterior segment optical coherence tomography (AS-OCT) and in vivo confocal microscopy (IVCM).
Material and methods
This was an interventional, case control study. Written informed consent was obtained from all patients before enrolment. The research adhered to the tenets of the Declaration of Helsinki and our institutional review board (Department of Medicine and Ageing Science of the University ‘G. d'Annunzio’ of Chieti-Pescara, Chieti, Italy) approved the project.
Forty-four consecutive Caucasian patients scheduled to undergo UCCC for refractory glaucoma were enrolled. Before enrolment, patients underwent best corrected visual acuity (BCVA), slit lamp biomicroscopy, Goldmann applanation tonometry (three measurements at 09:00, 12:00 and 16:00), fundus examination, and visual field (VF) test (30-2 test, full threshold, HFA II 750; Carl Zeiss Meditec, Inc, Dublin, California, USA).
Inclusion criteria were age ≥18 years, refractory glaucoma with at least one previous incisional surgery, uncontrolled IOP (>22 mm Hg) under maximal tolerated medical therapy (MTMT) and VF damage progression. MTMT required the use of all available classes of IOP lowering eye drops and oral acetazolamide, without significant side effects. VF damage progression was assessed with the trend-based analysis of the HFA Guided Progression Analysis software: when the magnitude of VF index slope was worse than 1% per year with a p value ≤0.05, the progression was considered clinically significant.
Exclusion criteria were history of topical or systemic therapy that could modify AH hydrodynamics, ciliary body trauma, surgical or laser procedure in the last 2 months, previous ciliary body ablation, retinal detachment, and pregnancy. If both eyes were eligible, the eye with the higher IOP or the more advanced glaucoma stage was included in the study.
Ten consecutive patients with medically controlled primary open-angle glaucoma (POAG), who did not undergo filtration surgery, were used as controls.
Inclusion criteria were mean IOP lower than 20 mm Hg with medical therapy unmodified in the last 6 months, and no history of VF progression during the last 12 months.
After enrolment, patients scheduled to surgery underwent ultrasound biomicroscopy (UBM; Aviso; Quantel Medical, Clermont-Ferrand, France), AS-OCT (RTVue XR Avanti; Optovue, Inc, Fremont, California, USA), and laser scanning IVCM (HRT III Rostock Cornea Module (RCM); Heidelberg Engineering GmbH, Dossenheim, Germany).
UCCC was considered successful when at least 30% reduction from preoperative IOP was obtained at 1 month follow-up. Eyes that met the above criteria and were not on supplemental anti-glaucoma medical therapy were defined as complete successes, whereas eyes that met the criteria with supplemental medical therapy were defined as qualified successes.19
The surgical procedure and the technical characteristics of the HIFU were previously described.14 Briefly, a 30 mm diameter ring containing six active piezoelectric elements was inserted in the upper part of a polymer-made coupling cone, previously placed in direct contact with the ocular surface, and filled with saline solution. The transducers are elliptic cylinder shaped segments of a 10.2 mm radius cylinder with a 4.5 mm width and a 7 mm length (active surface area of 35 mm2), distributed along the circumference of the ring. HIFU probes are commercialised in three different ring diameters (11, 12 and 13 mm), which allow them to fit most ocular sizes. UBM was used to select the right diameter of the probe, as previously described.14
Treatment consisted of the sequential activation of each transducer lasting 4 or 6 s. Patients randomly received (by computer-generated random numbers) a 4 s (group 1) or a 6 s (group 2) exposure time.
Postoperative treatment included topical dexamethasone and tobramycin (Tobradex; Alcon Laboratories, Inc, Fort Worth, Texas, USA) given for 2 weeks, three times a day. Preoperative hypotensive medications were maintained during the follow-up, including oral acetazolamide.
Follow-up visits included safety checks at day one and seven with BCVA determination, tonometry, slit lamp biomicroscopy and fundus examination. At day 30, mean IOP, VF, AS-OCT and IVCM were repeated. For controls, follow-up was scheduled at day 30, with AS-OCT, IVCM and IOP determination.
AS-OCT and IVCM examinations
AS-OCT and IVCM were performed to analyse the scleral and conjunctival features at the sites of insonification, before (day –1) and 30 days after the UCCC.
To avoid the sites of previous surgeries in the field of analysis we considered the transducers making contact with the superior-temporal or superior-nasal sectors. To comply with this intention we used methylene blue to mark the conjunctiva overlying the presumed site of transducer contact at 10 or 2 o'clock, delineating a 6×8 mm area (48 mm2), at least 3 mm away from the site of the previous surgery. To be sure that the transducer would match the marked conjunctival area, the coupling cone and the probe were also marked at 2 or 10 o'clock.
The AS-OCT and IVCM were performed within the marked area. A set of reference photographs was acquired at baseline to analyse the same area after 1 month.
In patients who underwent only one filtration surgery, we analysed the superior-temporal sector; in patients who received more than one surgery, we analysed the available unmanipulated sector.
For the AS-OCT, vertical cross-sectional scans were acquired to identify hyporeflective spaces (HS) within the sclera. The scan selection was set to the automatic mode so that video brightness and contrast were standard. A single operator (LB) performed all examinations and selected three high-quality images (from 10 randomly selected images). These images were evaluated and the results averaged by a second operator (ED). The mean intra-scleral hyporeflective spaces area (MIHSA, mm2) was calculated.
HS were defined as intra-stromal areas having a lower degree of reflectivity compared with the surrounding sclera. To distinguish between HS and physiological variations of the scleral homogeneity (and reflectivity), we considered only spaces with well defined edges and with a mean grey value lower than the 50% of the mean grey value of the surrounding sclera (figure 1). The reflectivity of normal sclera and HS was determined with Image J (http://rsb.info.nih.gov/nih-image). To calculate the reflectivity of the normal sclera, we selected three highly homogeneous stromal areas (100×100 µm) without evidence of HS in each of the three selected images. To be representative of the entire sclera, the three areas were selected in three different regions of the middle–outer sclera, where HS are normally rare.
The software determines the average grey value of the selected area, and this value corresponds to the sum of grey values of all pixels divided by the number of pixels. The mean value of the three areas was finally considered. All HS present in the image were analysed; the average grey value was determined within the entire area of each space.
IVCM was performed as previously described.20 Sequential images (400×400 µm) derived from automatic scans and manual frames were acquired at the intermediate layer of the epithelium. A single operator (VF) performed all examinations and selected eight high-quality images (from 40 randomly selected images), which were evaluated and the results averaged by a second IVCM operator (LT).
At month 1, reference photographs were carefully observed to re-analyse the same area: after recognising landmarks used at baseline, the conjunctival area was marked again with methylene blue, and examinations were performed giving particular attention to maintaining the RCM within the marked area.
IVCM was aimed at identifying epithelial microcysts, defined as round or oval-shaped optically clear structures, occasionally filled with amorphous material and roundish hyperreflective elements. The mean microcyst density (MMD, cysts/mm2) and area (MMA, µm2) were calculated. Microcyst density was calculated using the analysis software of the instrument, whereas microcyst area was determined using Image J. The AS-OCT and IVCM operators were masked for the patient surgical history and status.
MIHSA, MMD and MMA at day 30 were the primary outcomes; the IOP reduction was the secondary outcome. The relations between MIHSA, MMD, and MMA and IOP were investigated at month 1.
Analysis was performed using SPSS V.21.0 Software (SPSS Inc; Chicago, Illinois, USA). Student t and χ2 tests were used to evaluate age, IOP and gender differences, if appropriate, between groups, and between groups and controls. All data are reported as the mean±SD or frequency and percentage, as appropriate. Differences were considered statistically significant if the p value was less than 0.05. One-way ANOVA with Dunnett's correction was used to evaluate the differences in MIHSA, MMD, MMA and IOP between groups, and between groups and controls. Spearman's correlation analysis was used to investigate the relations between MIHSA, MMD, and MMA and IOP.
No significant differences were found between groups 1 and 2 for demographic and clinical data (table 1). Thirty patients (68%) were insonified with a 12 mm diameter probe, whereas 12 (28%) and two (4%) were insonified with a 13 and 11 mm probe, respectively. Twenty-four patients were treated with the 4 s dose (group 1) and 20 with the 6 s dose (group 2). UCCC was completed in all patients and none of them received a second treatment during the study period.
Baseline IOP was not significantly different between groups; at day 1, IOP significantly reduced in group 1, and at day 7, IOP reduced in both groups (p<0.001). At month 1, IOP significantly reduced in goth Groups (p<0.001), with group 2 showing values significantly lower compared with group 1 (p=0.001); in controls, IOP and mean number of medications did not significantly change compared with baseline (table 2). The overall success rate of UCCC was 63.63% (28/44), with a higher rate in group 2 (80%; 16/20) compared with group 1 (41.66%; 10/24) (p<0.05). All successful cases were classified as qualified success, and the number of medications significantly reduced compared with baseline in both groups (p<0.05) (table 2).
Overall, the procedure was safe without significant differences between groups for all reported complications (table 3). Groups 1 and 2 did not show significant baseline differences in MD (dB±SD) (−12.34±1.48 and −13.88±1.64, respectively) and BCVA (log MAR±SD) (0.92±0.15 and 0.93±0.18). MD and BCVA did not significantly change at month 1 (−12.84±1.74 and −13.61±1.54; 0.90±0.16 and 0.91±0.17). During the follow-up period none of the patients received systemic or topical therapy.
In all patients, the sclera presented a relatively homogeneous and hyper-reflective aspect with scattered intra-stromal HS (figures 2A, D and figure 3A). MIHSA was not significantly different between groups 1 and 2, and between groups and controls (table 4).
MIHSA significantly increased in both groups (p<0.05) with values higher in group 2 compared with group 1 (p<0.05) (table 4). Intra-scleral spaces appeared hyporeflective compared with the surrounding stroma, with different size and features. They were generally located in the middle and inner layers of the stroma, frequently surrounded by additional smaller spaces (figure 2B and E). In controls, MIHSA did not significantly change compared with baseline (table 4) (figure 3B). In one patient who received UCCC, a deep hyporeflective area was identified in the supra-choroidal space (figure 4).
MMD and MMA significantly increased in both groups (p<0.05) with values higher in group 2 (p<0.05) (figures 5 and 6B); in controls, MMD and MMA did not significantly differ compared with baseline (table 4).
Even though tissues outside the marked area were not investigated, in cases where scans were available, scleral and conjunctival changes were minimum or absent (figure 2C and F). A strong significant correlation was found between MIHSA (p=0.001; r=−0.967) and IOP in both groups.
UCCC is a cyclo-destructive procedure, which reduces the AH inflow by inducing a thermal necrosis of the ciliary epithelium.16 Additionally, a stimulation of the suprachoroidal and TS portions of the uveo-scleral outflow pathway has been documented.13–17
One month after UCCC, we observed a marked increase in the intra-scleral HS, which strongly correlated with IOP reduction.
We hypothesise that the increase in HS was a consequence of a scleral fibre delamination: in fact, a partial heating of supra-choroidal space, sclera and conjunctiva during the procedure is hypothesisable. The preliminary results of a thermal infrared imaging study (our unpublished data, 2015) supported this supposition: we observed a significant increase in the ocular surface temperature at the site of insonification, immediately after UCCC. Thus, the transducer produces a thermic halo (which is 1.89 mm3) with a temperature gradient from the ciliary body to the outer tissues. This mechanism may also account for the higher MIHSA increase in group 2, which received a prolonged duration of the insonification.
The increase in HS within the stroma may finally increase the hydraulic conductivity and the TS AH flow. This was stated by Denis and coworkers,14 who proposed that the exposure to ultrasound leads to microarchitecture changes in the uveo-scleral tract.
The effects of ultrasound on the sclera have also been reported in vitro, where the insonification of scleral samples induced a significant enhancement of the drug permeability (Investigation on cavitation contribution in scleral permeability enhancement. 13th International symposium of therapeutic ultrasounds; 12–15 May 2013, Shangai, Cina). By using electron microscopy, the authors observed less organised and loosely arranged tissues after insonification.
Also histology documented areas of scleral thinning and intra-stromal spaces within the thinned treated sclera after UCCC (Eye Tech Care material). The HS seen with AS-OCT were similar to intra-stromal spaces observed in histology. Though the tissue dehydration for the histological preparation could have affected the scleral architecture, the greater concentration of optically clear spaces at the site of insonification compared with the surrounding sclera confirmed a scleral effect of the procedure.
Though the procedure-related inflammation might play a role in the uveo-scleral outflow enhancement, no previous study described changes in the scleral anatomy in the presence of ocular inflammatory conditions or in patients taking prostaglandin analogues, such as described after UCCC. Moreover, the inflammatory effect after a cyclo-destructive procedure could be considered minimum or to have subsided after 30 days.21 ,22
The scleral thinning represents an effective procedure to enhance the TS AH outflow. Olsen et al23 found that the surgical thinning of the sclera induced a disorganisation of fibre bundles with widely spaced collagen fibres. Despite surgery and insonification thinning the sclera differently,14 scleral thinning positively affects the TS fluid movement. Though we did not observe scleral thinning, because AS-OCT cannot clearly show this modification, it is likely that such a change was present.
The increase in MMD and MMA confirmed the nature of AS-OCT findings since microcysts were proposed as signs of increased AH percolation through the sclera. Microcysts were first described in the bleb-wall epithelium of functioning trabeculectomy, as an indicator of trans-conjunctival AH percolation.24 ,25 Afterwards, they were also documented in the conjunctiva of medically treated patients with glaucoma, as a hallmark of TS AH outflow.26 ,27
In our study, the microcyst increase could be derived from an increased AH flow due to intra-scleral spaces and scleral thinning. This hypothesis was consistent with the microcyst increase observed after glaucoma surgeries producing scleral thinning, such as supra-choroidal shunt implantation and canaloplasty.20 ,28
Though we did not investigate the suprachoroid, since AS-OCT cannot easily image this site, in one case we observed HS in the suprachoroidal region. This aspect was consistent with an UBM study, which documented suprachoroidal spaces in more than 60% of UCCC.13
In our study UCCC had an overall success rate of 63.6% at 1 month, in line with the 66.7% reported by Denis et al.14 Also the higher IOP reduction in group 2 was consistent with the literature,13 ,14 which reported values ranging from 22.8% to 26.4% for the 4 s dose regimen, and from 28.2% to 38.2% for the 6 s dose.
The present study has some limitations. First, cases and controls differ because controls did not have refractory glaucoma. However, the inclusion of patients with uncontrolled refractory glaucoma as a control group would have raised ethical concerns, since they would have not received treatment before the end of the study. However, cases and controls present the same baseline AS-OCT and confocal parameters, which rule out crucial differences between patients. In addition, the glaucoma type and/or the presence of a progressing disease do not induce anatomic differences in the conjunctiva and the sclera. The only factor that could have modified scleral and conjunctival anatomy is the surgical procedure. Nevertheless, examinations were performed at a distance from the site of previous surgery, in unmanipulated tissue. Second, our results do not permit the impact of the TS outflow in the final IOP reduction to be determined. Nevertheless, the strong correlation between MIHSA and IOP indicated that scleral modifications play a crucial role. Third, AS-OCT does not allow the ciliary body to be studied; thus, we cannot clarify whether successful and unsuccessful cases derived from more or less effective ciliary body coagulation. Fourth, IVCM and AS-OCT may present a significant level of subjectivity, even for an experienced operator. Though we did not evaluate the intra-subject agreement, previous studies reported excellent intra-individual and inter-individual variability for both instruments.29 ,30 Finally, since microcysts can also be found in healthy subjects and in some ocular surface diseases, their significance should be clarified. However, several studies indicated that in glaucoma they are a hallmark of AH outflow.20 ,25–28
In summary, though the main mechanism for IOP reduction after UCCC is a decrease in the AH inflow, the increase in the TS AH outflow seems to play a critical role in the final success. Further studies measuring the TS outflow with more accurate methodologies are mandatory to confirm our preliminary data. In addition, probe modification allowing a selective scleral insonification should be considered in the near future.
Finally, while this study specifically analysed the impact of the procedure on the uveo-scleral outflow in the first postoperative period (when modifications are much more pronounced), a further long-term follow-up study should be performed to evaluate if these modifications persist over time.
RM and LA contributed equally to this work and share primary authorship.
Contributors RM: writing the article; critical revision of the article. LA: conception and design; writing the article; critical revision of the article. VF: provision of patients; performing examinations; statistical contribution. LT: writing the article; performing examinations. LB: provision of patients; performing examinations; data collection. SDS: literature research; data collection. ED: literature research; critical revision of the article. GM: critical revision and final approval of the article.
Competing interests None declared.
Patient consent Obtained.
Ethics approval Institutional Review Board.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement The present study contributed to clarifying the mechanism of action of ultrasonic cyclocoagulation in patients with refractory glaucoma. Particularly, we documented a scleral architecture rearrangement, which enhances trans-scleral aqueous humour drainage.