Pascal Short Pulse plus Subthreshold Endpoint Management Laser for Diabetic Macular Edema: The “Sandwich” Technique Keywords: Pascal laser, short pulse laser, endpoint management, diabetic macular edema.

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ABSTRACT (word count: 282)

Purpose: To evaluate the effect of combined sublethal laser modalities, short pulse photostimulation plus endpoint management subthreshold LASER (the Sandwich technique: SWiT), on central subfield thickness (CST) of patients with diabetic macular edema (DME).

Methods: This study was conducted in eyes with DME in real-life conditions and represents a consecutive retrospective analysis of 37 patients with center-involving DME that were treated with the SWiT laser. The technique consisted of a mean number of 200 (range number: 95-600) short pulse duration (SPD) LASER spots performed on juxta- and perifoveal area (within 6mm ETDRS grid) 500µm away from the foveal center and 500 (range number: 300-800) subthreshold endpoint management (EpM) laser spots performed juxta- and perifoveal area (within 6mm ETDRS grid) 300µm away from the foveal center. All patients underwent ophthalmological evaluations that included ETDRS BCVA and CST measurement on spectral domain optical coherence tomography (SD-OCT) before and after SWiT-laser therapy. The mean follow-up time was 19.2 months (range: 2-60 months).

Results: 35 out of 37 cases had an improvement in CSFT and BCVA following treatment. At baseline, mean CST (µm) ± standard error (SE) and mean BCVA (log-MAR) ± SE was 456.95 ± 37.00 and 0.71 ± 0.29 respectively. After a mean follow-up of 19.2 months, mean CSFT (µm) ± SE and BCVA (log-MAR) ± SE were 272.09 ± 9.10 (p < 0.0001) and 0.54 ± 0.26 (p = 0.003). A statistically significant reduction on CSFT (µm) and improvement on BCVA (log-MAR) was noted compared to baseline. The mean number of anti-VEGF intravitreous injection during this period was 3 in 6 patients.

Conclusion: The novel SWiT laser therapy reduced CSFT and improved BCVA in this retrospective case series. Further prospective studies are warranted.

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INTRODUCTION

In the next three decades, the prevalence of diabetes will more than triple universally, radically increasing the

worry of this disease worldwide.1 Diabetic macular edema (DME) is the main cause of visual impairment in diabetic patients.2-4 Formerly, in the 80’s the Early Treatment Diabetic Retinopathy Study (ETDRS) revealed a substantial advantage of laser photocoagulation for the treatment of clinically significant DME, diminishing the incidence of visual

loss by approximately 50% at 3 years’ follow-up.5

In 2002, a National Eye Institute-sponsored collaborative network, the Diabetic Retinopathy Clinical Research Network (DRCR.net), compared steroid versus laser treatment for DME. Of note, 26% of DME patients treated with laser did gain 15 or more letters of vision at three years.6 Regardless reasonable results previously to anti-VEGF era, the original ETDRS photocoagulation technique raised many thoughts about adverse events entailing experts progressively improve the technique over the years. Therefore, a wide consensus regards the modified (m)ETDRS laser protocol, as standard modality in main trials from DRCR.net was adopted based on specialist’s survey.7 Despite of the refine techniques, adverse events such as central scotoma, loss of central vision, and decreased color vision can still happen.

Vascular endothelium growth factor (VEGF), originally identified as vascular permeability factor, is a potent inducer of vessel permeability and macular edema.8 Recent trials using anti-VEGF therapies for DME have shown significant results. Despite the use of anti-VEGF therapy as first-line treatment for centrally involved DME, there are many patients whose response to therapy is poor or transient at best. In the RISE/RIDE trials, approximately 50% of patients failed to achieve ≥ 15-letter gain in BCVA despite 2 years of monthly ranibizumab (0.5 mg) injections.9

Additionally, some of these patients suffer from persistent or worsening edema and/ or vision loss despite treatment.10,11

In 2010, the DRCR.net (protocol I) showed that in the one year follow up of this study, 64% of patients receiving intravitreous ranibizumab injection (IVR) plus prompt laser and 52% of patients receiving ranibizumab plus deferred laser group met “success” criteria (VA letter score ≥84 [≥20/20] or CST < 250µm) and did not receive any injection.12 Only 32% of patients in the IVR plus prompt laser group and 21% of participants in the IVR plus deferred laser group had no IVR injections between the 1- and 2-year visit.12 In addition, patients that received prompt laser required a mean of 13 IVR injections, while patients that did not underwent laser (deferred laser group) required 17 IVR injections during the 5-year follow-up13,14

The noteworthy DRCR.net study-protocol T demonstrated that aflibercept therapy had superior outcomes in eyes with worse baseline acuity, however the laser rescue therapy was required for persistent DME defined based on protocol-specified criteria.15 Remarkably, over 2 years 41, 64 and 52% of eyes treated with aflibercept, bevacizumab or ranibizumab, respectively, received focal/grid laser (P≤0.01 for each pairwise comparison).16 Intriguingly, this protocol revealed high percentage of laser rescue therapy pondering thoughts as a combined treatment study instead of single drugs regimen comparison.

Refined skills have emerged desiring low-intensity photostimulation with barely visible titration, short time release, and high-density delivery. The SPD and subthreshold laser modalities (e.g., micropulse diodo-laser or Pascal®- EpM) allow an effective therapy with only sublethal thermal elevations, avoiding the excessive heat that causes visible burns, tissue necrosis, and related collateral effects.17-19 Our group proposed a combined Pascal laser modalities: SPD plus EpM as first described on literature and named “Sandwich Technique” intending to add a flexible repair strategy on DME handling.

METHODS

Study design

This study adheres to the Declaration of Helsinki, and was approved by the Ethics and Research Committee of Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Univerisidade de São Paulo. Data were collected in a retrospective fashion. The records of all patients in a public vitreoretinal subspecialty practice who had undergone

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SWiT laser therapy for the primary diagnoses of DME were reviewed. This included 37 eyes from April 2009 to January 2019.

Patient demographics

We assessed the parameters of patient age, sex, hemoglobin A1c, hypertension (HTN) history, diabetes long-term history, previous pan-retinal photocoagulation (PRP), and diabetic retinopathy (DR) severity (table 1).

Table 1: Baseline characteristics & demographic data

Examination Procedures

All patients underwent a comprehensive ophthalmic examination comprising BCVA with an ETDRS vision chart, slit-lamp biomicroscopy, indirect ophthalmoscopy, near-infrared reflectance scanning laser ophthalmoscopy, fluorescein angiography, and SD-OCT (Spectralis; Heidelberg Engineering).

Patient eligibility and exclusion criteria

Inclusion criteria were as follows: Eligible participants were aged 18 years with diabetes mellitus (type 1 or 2); have a BCVA in the involved eye between 0.3 logMAR (Snellen equivalent: 20/32) and 1.3 logMAR (Snellen equivalent: 20/400); DME with CSFT > 300 µm on SB-OCT, despite of macular laser photocoagulation, cataract surgery and intraocular injection performed at least 4 months previously. Exclusion criteria: any evidence of vitreomacular traction on SD-OCT; no history of vitreoretinal surgery; no macular capillary dropout on fluorescein angiography.

Treatment regimen

All treatments were provided with Pascal® photocoagulation unit, which uses a 532-nm frequency doubled solid-state green laser source. An expert physician performed the laser therapy into all patients. The SWiT laser treatments were administered in a grid-SPD (diffuse edema) or focal-SPD single-shot (mainly to extra-foveal edema) with an association of subthreshold EpM software (a computational model–based algorithm providing adjustment of laser power and duration) to treat the areas of thickening guided by thickness map on SB-OCT imaging. Retinal microaneurysms were not treated focally. The SWiT laser therapy involved both photo thermal stimulation methods: SPD and subthreshold EpM, and got applied simultaneously for all cases. Briefly, in this novel technique we first

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applied the SPD titration with single shots laser duration of 10 milliseconds, spot size was 100 µm and power was adjusted to give a light gray barely visible laser lesion tested besides arcades inward on posterior pole. Subsequently, to treat extra-foveal DME fewer focal SPD single-shots were applied to reach thickened area guided by thickness map till 500 µm from foveal center. Also, to treat diffuse foveal DME, a grid-SPD with multiple simultaneous shots were applied using 1.200 radius over thickened area till 500 µm from foveal center. Besides, subthreshold EpM with 200µm spot size and lowering energy to 30% was associated to reach nearer juxta-foveal area till 300µm from foveal center and overlapped within grid-SPD treated areas. The subthreshold EPM laser spots were placed in a confluent grid 0.25 spot diameters apart (figure 1).20,21

Figure 1: Schematic representation of laser display and anatomical responsiveness.

Statistical analysis

Data were collected in a retrospective fashion from chart review. Statistical analysis was undertaken using a single-arm univariate Student t-test with statistical significance corresponding to a P value of 0.05. Statistical analyses were performed using JMP 10.0.0 (2010; SAS Institute Inc, Cary, North Carolina, USA) software. Mean BCVA and CSFT measured after SWiT laser therapy were compared with baseline BCVA and CSFT mean, which were performed using multiple analysis of variance (MANOVA) for repeated measurements.

RESULTS

Between April 2009 and January 2019, 37 subjects (mean age 65.8 ± 8.2 years; 50% women) were enrolled (figures 2-4, example cases). A fraction of 90.5% subjects of total had a minimal of 5 months’ follow-up after SWiT laser therapy. The average of total follow-up period was 19.2 months. Two new cases (5.4%) had recently diagnosed DME (< 6 months) and were performed SWiT laser therapy as a first option treatment. Most of subjects (94.6%) were considered chronic DME status (> 6 months’ duration) and 24.6% of that had never attained any intravitreous injection and/or laser procedure instead of diet orientation. Chronic cases (70%) had already received different DME therapies (anti-VEGF and/or TAAC and/or ETDRS laser) performed at least 4 months previously. Eight current treated cases (21.6%) have changed prn-IVR therapy and switched to SWiT laser therapy as they got unsatisfactory response. Three cases (16,2%) required at least one IVR as needed after 3 months forward SWiT laser throughout 18 months’ follow-up.

Figure 2: A patient with chronic DME and recurrent edema.

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Figure 3: A patient with chronic DME and recently not sustained anti-VEGF response. (

Figure 4: A patient with chronic DME and recently not sustained anti-VEGF response.

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Effect of Treatment on Retinal Thickening (figure 5A)

Central subfield thickness decreased over mean 19.2 (months) period compared to baseline (P< 0.0001). At baseline, mean CST (µm) ± SE was 456.95 ± 37.00 and decreased to 272.09 ± 9.10 forward SWiT laser therapy. Most of eyes (77.7%) had CST decreased about more than 100µm at least.

Figure 5A: mean BCVA (logMAR) after SWiT laser compared to baseline.

Effect of Treatment on Visual Acuity (figure 5B)

At baseline, mean BCVA (log-MAR) ± SE was 0.71 ± 0.29 (Snellen equivalent: 20/100). After mean follow-up

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of 19.2 months, mean BCVA (log-MAR) ± SE were 0.54 ± 0.26 (Snellen equivalent: 20/70). A statistically significant improvement on BCVA (log-MAR) was noted compared to baseline (p = 0.003). One third (30%) of the subjects had no improvement of BCVA from baseline. In counterpart, 70% had BCVA improvement about more than 5 letters. at least.

Figure 5B: mean CSFT (µm) after SWiT laser compared to baseline.

The t-test and single variable correlation between BCVA and CSFT at baseline and after SWiT laser therapy

Of note, there is statistically significant difference before and after SWiT laser therapy for the both variables: BCVA and CST. Also, there is a significant correlation relationship (r = 0.64) between BCVA previous SWiT laser therapy and BCVA after SWiT laser therapy. Furthermore, there is a significant correlation relationship ( r = 0.52) between CSFT after SWiT laser therapy and BCVA after SWiT laser therapy. Multivariable analysis corroborates that only 2 factors (CST after and BCVA previous SWiT laser therapy) influence in the BCVA after SWiT laser therapy. Therefore, CSFT previous SWiT laser therapy is not significant correlated to the BCVA after SWiT laser therapy.

DISCUSSION

The sandwich technique received this name because there is an outer ring of SPD treatment involving an inner ring of lower intensity laser EpM, resembling a sandwich (figure1A-B). We obviously decided to apply a more intense laser (SPD) in the outer ring, farther from the foveal center, while subthreshold laser (EpM) was painted more intensive nearly to foveola. In fact, with sublethal low-intensity threshold technique (i.e.; SPD) that produce only low and confined thermal elevations, there is very little lateral spread of heat from the RPE spots directly targeted by the laser. In theory, clinical response could be related to the laser-induced activation of cytokine expression and upregulation of

matrix metalloproteinase (MMP)-3 in the retina, promoting subsequent cellular changes that correct the pathologic imbalance and stop the spread and migration of the RPE cells at the edges of the lasered site. 22-25. Previously determined, enhanced expression of the heat shock protein (HSP- 70) in the retina begins at laser power approximately half of the RPE damage threshold.26 Once laser retinal interactions trigger HSP activation, an initiation step in a cascade of reparative phenomena improves RPE function, retinal autoregulation, reparative acute inflammation, reduced markers of chronic inflammation, and immunomodulation (figure 1C).57,58 Idealized subthreshold photocoagulation (i.e., EpM) is defined as photocoagulation resulting in laser spots that are not visible under any light microscope.29,30 It has been

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suggested that subthreshold photocoagulation helps to increase the permeability of RPE in the early stage, and promotes the discharge of effusion in areas of edema. In the later stage, RPE repair promotes metabolism and water discharge from the retina to the choroid.31,32 Moreover, a lowest risk level of damage to the RNFL and lower inflammatory reactions with visual function preservation is guaranteed with this technique.33 Some clinical studies have concluded that micropulse diode laser subthreshold photocoagulation could improve visual acuity and alleviate DME.34

Despite of lack of studies comparison between subthreshold low-energy micropulse exposures (exhaustively studied since 90’s)35 and subthreshold Pascal-EpM modern technique, it can be hypothesized theoretically that both nondestructive regimens may have analogous efficiency as an nondamaging photostimulation laser modalities. At this point, is relevant clarify that SPD (10 milliseconds duration) at 100% energy levels (threshold barely visible), the photoreceptors and RPE are selectively target, leaving the inner retina intact. These lesions heal over 2 months, reestablishing the photoreceptors layer and local synapses between migrated photoreceptors and preserved bipolar cells.36 Momentary disruption of the ellipsoid band suggests that each hypereflective vertical mark may consist of coagulated photoreceptor elements and Muller cells within the OPL (figure 1A-1B). Localized proliferation within the

apical RPE with no morphological alterations within inner retina correlates with reported histopathological work. 37

Over time, lesions created by shorter duration laser tended to contract instead of expand38 (as do lesions created with longer pulse durations, i.e., figure 1B: white dashed square). Evidence of treatment effect by short term duration for the treatment of DME has been published.39Also, with the advent of the Pascal laser, multiple barely visible laser lesions can be applied safely and with decreased risk of retreating a particular area.40

This real-life study demonstrated a statistically significant reduction of CST (p<0.0001) and significantly improvement of BCVA (p=0.003) after SWiT laser for DME. The results of the present study highlight the importance of sublethal laser modalities in managing DME. Pei-Pei et al.41 showed results from prospective randomized study comparing two Pascal laser modalities: subthreshold (EpM) versus threshold (SPD) laser therapy. There were not significantly difference between the groups on BCVA (p=0.428) and CST (p=0.399) outcomes at 6 months’ follow-up. Both groups demonstrated significantly difference on mean CST at six months follow-up compared to baseline (p<0.05). However, at 6-month the threshold-group demonstrated no significantly difference on mean BCVA compared to baseline (p=0.065), but the subthreshold-group showed signifincatly difference on mean BCVA compared to baseline (p=0.035),

The absence and slight presence of thermal spread from subthreshold EpM and threshold barely visible SPD respectively, should be compensated for with a higher density of applications to effectively treat thickened OCT guided areas. The density optimization strategy plays a key role as a flexible repair plan for the treatment of DME under subvisible laser protocols.42 Structure restoration on CSFT map is molded according thicker distribution so that diverse anatomical form of edema is treatable with individual density allocation allowed by SWiT laser approach. Thinner lately DME (<400µm) with less disorganized retinal layers and better BCVA (>20/50 Snellen) suggests favorable response to less density of SPD shots but higher subthreshold EpM inversely (figure 2). In opposite, thicker longstanding DME with haphazard full retinal layers and worse BCVA (<20/50 Snellen) may implies better response to high density of joined modalities: SPD and EpM. We believe that higher rates of threshold-SPD in unhealthier cells status, potentially forces lasting incompetent RPE/Glial cells to survive (“defibrillation effect”) or stimulate surrounding viable cells for photoreceptor rearrangement and/or migration. While, less functional and structural suffered retinal cells or even adjacent regular RPE cells may be more suitable to high proportion of subthreshold-EpM for effectively alleviating DME.

The present study has some design limitations as a retrospective study, including a relatively small sample size and lack of macular visual function examination such as contrast sensitivity and micro-perimetry . In addition, a more extensive prospective and randomised study protocol will provide further valuable insight into the effectiveness and reliability of this new strategy explored by photorestauration SWiT laser. Lack of trials using a sophisticated technique still scant in the researcher community. Difficulties as the high investment devices, extensively physician training and time consuming procedure discourage everyone’s. Discerning, an ideally moment to offer appropriate approach is

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essentially reliant on various considerations including the previous anti-VEGF time course response, associated ocular pathologies (i.e., glaucoma), past of PPV, anatomical macular status (i.e., VMT and MER), capillary perfusion functioning (i.e., ischemic maculopathy), clinical conditions (i.e., pregnancy, recent cardiac infarction and cerebral vascular accident) and aging DME. In good hands, inherent advantages from appropriated laser technique can prevent the excessive number of intravitreous injections, particularly regarding patient convenience and treatment cost effectiveness. The proposed SWiT laser therapy as a photorestoration may offer a promising alternative for an enhanced beneficial effectiveness, putting in check the ancient mETDRS photocoagulation protocol.

Until nowadays, even with the advent of sophisticated drugs and protocols there is a recognition that laser as associated therapy still needed for many DME cases and might demand less costs, according long term follow-up. Beyond efficacy and safety, an important public health consideration when comparing intravitreous anti-VEGF therapies for DME is their difference in cost; $1850 for 2.0 mg of aflibercept, $60 for 1.25 mg of repackaged bevacizumab and $1170 for 0.3 mg of ranibizumab.43

Therefore, in parallel to the renovated and costly anti-VEGF studies, whereas less exploited in the research community the retinal laser therapy has been appreciated and required in many clinical trials protocols. A recent review, published clinically relevant findings from 2 years DRCR.net Protocol T and potential future directions for persistent DME were taking in debate.44 Refine the role of focal/grid laser as a supplement to anti-VEGF for DME management was one of issues in focusing. As we know, traditionally the (m)ETDRS approach still currently suggested in most well-known DME trials, but not encouraged by the laser experts presently.

Avoiding those archaic techniques that includes thermal retinal injury photocoagulation, specialists have published advanced practices aimed maximizing clinical effectiveness and allowing a finer control of the photothermal effects induced at the level of the RPE.45,46

This retrospective study take in account diverse real-life cases and the idea to combine refine sublethal techniques as the novel SWiT laser method, revealead promising results in several types of DME worthwhile future prospective studies.

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Figure 1: Schematic representation of laser display and anatomical responsiveness. (A) SB-OCT image of retinal edema prior to SWiT laser therapy and representative image (white dashed) of photothermal effects producing retinal photoreparation. (B) SB-OCT image of solved macular edema showing SWiT arrangement comprising barely visible marks spreads 500µm from foveal center (green; SPD treated area) and invisible

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marks (purple; EpM treated area) overlapping the SPD treated area and reaching till 300µm from foveal center. Besides, in the white dashed area a representative image of ETDRS laser scar performed in the past. (C) Representative image of RPE cell thermal activation and intranuclear heat shock fator (HSF) stimulation to generate HSPs with subsequently anti-apoptotic factors formation and inflammation factors depletion.Figure 2: A patient with chronic DME and recurrent edema. (A) CSFT map overlying near-IR image reveal diffuse center involved macular edema. (B) Late phase of fluorescein angiography showing cystoid hyperfluorescent leaking (red dashed square). (C) Baseline SB-OCT demonstrating hypo reflectivity cysts and atrophic lesions temporally to the fovea from previous ETDRS laser therapy (yellow arrowhead). The CSFT is 424µm and illustrative laser target (green arrow; SPD and pink stars; EPM). (D) Five months after SWiT laser therapy with CSFT map adequately. (E) Late phase of fluorescein angiography without leaking (red dashed square) and presence of tiny hypofluorescent dots spread widely on macular area, as a result to SPD laser array (green arrowhead). Representative invisible EPM laser target (pink star). (F) SB-OCT image of five months solved macular edema showing hiperreflective dots in the outer retina because of photothermal cells activation from SPD laser pattern (green asterisk). The CSFT impressively decreased to 294µm. (G) Eighteen months later, the SB-OCT showed stable CSFT of 287µm.Figure 3: A patient with chronic DME and recently not sustained anti-VEGF response. (A) SB-OCT monthly follow-up of prn-IVR therapy and switched therapy to SWiT laser on week 16, after two consecutive unresponsive IVR injections. (B) Baseline intermediate phase of fluorescein angiography (orange dashed square) revealing hyperfluorescent microaneurisms (red arrowhead) correlated to thickened edema (cyan dashed square) on thickness-map. SB-OCT image with CSFT of 304µm on baseline. (C) Intermediate phase of fluorescein angiography (yellow dashed square) of twenty-week follow-up showing hypofluorescent density spots (green arrowhead) related to SPD laser shots. Absence of edema (cyan dashed square) on thickness map and inactive microaneurisms undetectable. SB-OCT image with CSFT decreased to 255µm forward 4 weeks of SWiT therapy.Figure 4: A patient with chronic DME and recently not sustained anti-VEGF response. SB-OCT monthly follow-up of prn-IVR therapy and switched therapy to SWiT laser on week 24, after two consecutive unresponsive IVR injections. (B) Baseline thickness map showing CSFT of 743µm (red dashed square) and cystic hyper-reflectance edema on near-IR (cyan dashed square). (C) 24 th week follow-up, showing CSFT of 818µm on thickness map (green dashed square) and near-IR revealing hyper-reflectance dots (cyan dashed square) from just made SWiT laser (green and pink asterisks of SPD and EPM respectively). (D) SB-OCT image with CSFT decreased to 217µm forward 8 weeks of SWiT therapy (yellow dashed square) and absent cystic hyper-reflectance edema on near-IR (cyan dashed square).

Figure 5A: mean BCVA (logMAR) after SWiT laser compared to baseline.

Figure 5B: mean CSFT (µm) after SWiT laser compared to baseline.

Table 1: Baseline characteristics & demographic data

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