Investigation of the therapeutic mechanism of subthreshold micropulse laser irradiation in retina.

PURPOSE: Subthreshold micropulse laser irradiation has been used for the treatment of retinal edema; however, there are few reports about the mechanism of its therapeutic effect. In this study, we compared threshold short pulse and subthreshold micropulse laser irradiation in mice and investigated their mechanism. METHODS: Nine to 12-week-old male C57BL/6J mice were used in this study. After general anesthesia, threshold short pulse or subthreshold micropulse laser irradiation was performed on the right eye using IQ577. Enucleation was performed 24 h after the laser irradiation, and histological and gene expression analyses were carried out. RESULTS: Coagulation spots and atrophy of the retinal pigment epithelium were observed after threshold short pulse laser irradiation but not after subthreshold micropulse laser irradiation. Twenty-four hours after laser, aquaporin (AQP) 1, 2, 7, and 11 levels were significantly elevated by 1.7- to 3-fold in the threshold short pulse laser group compared with non-treated control group. AQP 3 was increased significantly and prominently by 100-fold. VEGF-A and VEGFR2 were upregulated 1.5- and 2.3-fold, respectively. In the subthreshold micropulse laser group, AQP 3 was increased by 6-fold compared with the non-treated control group. Angiopoietin-1 and the adrenomedullin (AM) receptor CLR were decreased by 0.6-fold and 0.5-fold, respectively. CONCLUSION: Threshold short pulse laser irradiation caused retinal damage and prominent changes in the expression of various genes. Contrarily, subthreshold micropulse laser irradiation did not induce retinal damage; it upregulated AQP 3, which might have improved retinal edema by drainage of subretinal fluid.

Surgical Approaches for Cell Therapeutics Delivery to the Retinal Pigment Epithelium and Retina.

Developing successful surgical strategies to deliver cell therapeutics to the back of the eye is an essential pillar to success for stem cell-based applications in blinding retinal diseases. Within this chapter, we have attempted to gather all key considerations during preclinical animal trials.Guidance is provided for choices on animal models, options for immunosuppression, as well as anesthesia. Subsequently we cover surgical strategies for RPE graft delivery, both as suspension as well as in monolayers in small rodents, rabbits, pigs, and nonhuman primate. A detailed account is given in particular on animal variations in vitrectomy and subretinal surgery, which requires a considerable learning curve, when transiting from human to animal. In turn, however, many essential subretinal implantation techniques in large-eyed animals are directly transferrable to human clinical trial protocols.A dedicated subchapter on photoreceptor replacement provides insights on preparation of suspension as well as sheet grafts, to subsequently outline the basics of subretinal delivery via both the transscleral and transvitreal route. In closing, a future outlook on vision restoration through retinal cell-based therapeutics is presented.

An easy, rapid method to isolate RPE cell protein from the mouse eye.

The retinal pigment epithelium (RPE) is essential for maintaining the health of the neural retina. RPE cell dysfunction plays a critical role in many common blinding diseases including age-related macular degeneration (AMD), diabetic retinopathy, retinal dystrophies. Mouse models of ocular disease are commonly used to study these blinding diseases. Since isolating the RPE from the choroid has been challenging, most techniques separate the RPE from the retina, but not the choroid. As a result, the protein signature actually represents a heterogeneous population of cells that may not accurately represent the RPE response. Herein, we describe a method for separating proteins from the RPE that is free from retinal and choroidal contamination. After removing the anterior segment and retina from enucleated mouse eyes, protein from the RPE was extracted separately from the choroid by incubating the posterior eyecup with a protein lysis buffer for 10 min. Western blot analysis identified RPE65, an RPE specific protein in the RPE lysates, but not in choroidal lysates. The RPE lysates were devoid of rhodopsin and collagen VI, which are abundant in the retina and choroid, respectively. This technique will be very helpful for measuring the protein signal from the RPE without retinal or choroidal contamination.

Morphologic changes in the retina after selective retina therapy.

PURPOSE: To investigate structural changes in the retina by histologic evaluation and in vivo spectral domain optical coherence tomography (SD-OCT) following selective retina therapy (SRT) controlled by optical feedback techniques (OFT). METHODS: SRT was applied to 12 eyes of Dutch Belted rabbits. Retinal changes were assessed based on fundus photography, fluorescein angiography (FAG), SD-OCT, light microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) at each of the following time points: 1 h, and 1, 3, 7, 14 and 28 days after SRT. BrdU (5'-bromo-2'-deoxy-uridine) incorporation assay was also conducted to evaluate potential proliferation of RPE cells. RESULTS: SRT lesions at1 h after SRT were ophthalmoscopically invisible. FAG showed leakage in areas corresponding to SRT lesions, and hyperfluorescence disappeared after 7 days. SD-OCT showed that decreased reflectivity corresponding to RPE damage was restored to normal over time in SRT lesions. Histologic analysis revealed that the damage in SRT lesions was primarily limited to the retinal pigment epithelium (RPE) and the outer segments of the photoreceptors. SEM and TEM showed RPE cell migration by day 3 after SRT, and restoration of the RPE monolayer with microvilli by 1 week after SRT. At 14 and 28 days, ultrastructures of the RPE, including the microvilli and tight junctions, were completely restored. The outer segments of the photoreceptors also recovered without sequelae. Interdigitation between the RPE and photoreceptors was observed. BrdU incorporation assay revealed proliferation of RPE on day 3 after SRT, and peak proliferation was observed on day 7 after SRT. CONCLUSION: Based on multimodal imaging and histologic assessment, our findings demonstrate that SRT with OFT could selectively target the RPE without damaging the neurosensory retina. Therefore, the use of SRT with OFT opens the door to the possibility of clinical trials of well-defined invisible and nondestructive retina therapy, especially for macular disease.

Thermofusion of the retina with the RPE to seal tears during retinal detachment repair.

PURPOSE: To develop an animal model to test the hypothesis that immediate adhesion of the retina to the choroid (retinopexy) can be created by elimination of the water separating the retina from the retinal pigment epithelium (RPE) prior to photocoagulation. The retina and RPE are hydrophobic lipoprotein structures separated intraoperatively by a thin layer of fluid despite surgical drainage. If the RPE and retina are contacting, heating should create a unified local coagulum and achieve instantaneous fusing of the retina and RPE, thus sealing the subretinal space around the retinal tear. The surgical technique and histological findings in a rabbit model of rhegmatogenous retinal detachment (RRD) are reported here. METHOD: Nine Dutch-belted, pigmented rabbits underwent vitrectomy with lensectomy, creation of localised retinal detachment by subretinal injection of balanced salt solution (BSS), enlargement of the hole and fluid-gas exchange to "re-attach" the retina. Dehydration of the retina surrounding the hole was achieved by an airstream from a flute needle. A laser (810 nm) was applied in long pulses to achieve a mild retinal reaction around the hole in the dehydrated adjacent retina. The BSS irrigation was resumed. Eyes were then enucleated and the treated retina examined histologically. RESULTS: The dehydrated and lasered retinal tear margin demonstrated fusion of the retina with the RPE/choroid. The non-dehydrated adjacent areas showed thermal tissue changes in the retina, RPE/choroid and adjacent sclera but remained separated by persistent subretinal fluid and no fusion or unified coagulum developed. CONCLUSION: Immediate laser-induced thermal fusion of the retina with the RPE at the margin of a retinal tear can be achieved by removing the subretinal fluid prior to photocoagulation. The integrated coagulum seals the tear margin preventing further fluid entering the subretinal space, thus correcting the cause of RRD. This method may facilitate RRD repair without buckling or internal tamponade.

Protective effect of a laser-induced sub-lethal temperature rise on RPE cells from oxidative stress.

Recently introduced new technologies that enable temperature-controlled laser irradiation on the RPE allowed us to investigate temperature-resolved RPE cell responses. In this study we aimed primarily to establish an experimental setup that can realize laser irradiation on RPE cell culture with the similar temperature distribution as in the clinical application, with a precise time/temperature history. With this setup, we conducted investigations to elucidate the temperature-dependent RPE cell biochemical responses and the effect of transient hyperthermia on the responses of RPE cells to the secondary-exposed oxidative stress. Porcine RPE cells cultivated in a culture dish (inner diameter = 30 mm) with culture medium were used, on which laser radiation (λ = 1940 nm, spot diameter = 30 mm) over 10 s was applied as a heat source. The irradiation provides a radially decreasing temperature profile which is close to a Gaussian shape with the highest temperature in the center. Power setting for irradiation was determined such that the peak temperature (Tmax) in the center of the laser spot at the cells reaches from 40 °C to 58 °C (40, 43, 46, 50, 58 °C). Cell viability was investigated with ethidium homodimer III staining at the time points of 3 and 24 h following laser irradiation. Twenty four hours after laser irradiation the cells were exposed to hydrogen peroxide (H2O2) for 5 h, followed by the measurement of intracellular glutathione, intracellular 4-hydroxynonenal (HNE) protein adducts, and secreted vascular endothelial growth factor (VEGF). The mean temperature threshold for RPE cell death after 3 h was found to be around 52 °C, and for 24 h around 50 °C with the current irradiation setting. A sub-lethal preconditioning on Tmax = 43 °C significantly induced the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio, and decreased H2O2-induced increase of intracellular 4-HNE protein adducts. Although sub-lethal hyperthermia (Tmax = 40 °C, 43 °C, and 46 °C) caused a slight increase of VEGF secretion in 6 h directly following irradiation, secondary exposed H2O2-induced VEGF secretion was significantly reduced in the sub-lethally preheated groups, where the largest effect was seen following the irradiation with Tmax = 43 °C. In summary, the current results suggest that sub-lethal thermal laser irradiation on the RPE at Tmax = 43 °C for 10 s enhances cell defense system against oxidative stress, with increasing the GSH/GSSG ratio. Together with the results that the decreased amount of H2O2-induced 4-HNE in sub-lethally preheated RPE cells was accompanied by the lower secretion of VEGF, it is also strongly suggested that the sub-lethal hyperthermia may modify RPE cell functionality to protect RPE cells from oxidative stress and associated functional decrease, which are considered to play a significant role in the pathogenesis of age-related macular degeneration and other chorioretinal degenerative diseases.

Functional recovery after experimental RPE debridement, mfERG studies in a porcine model.

BACKGROUND: The correlation between histologically identified regeneration of retinal pigment epithelium (RPE) and functional outcome measured by multifocal electroretinography (mfERG) following surgical debridement is examined in a porcine model. In humans, visual acuity is reduced in diseases with RPE loss such as RPE tears and geographic atrophy. Hypopigmented RPE is known to cover the lesion after RPE debridement in the pig, but it is unclear whether this leads to a return of photoreceptor function. METHODS: RPE debridement was performed in ten pigs by vitrectomy and retinotomy, and by brushing the Bruch's membrane with a silicone catheter. Immediately following surgery (baseline) and after 2 and 6 weeks respectively, the animals were examined by mfERG, fundus photographs (FPs), fluorescein angiograms (FAs), and histopathology. RESULTS: The mfERG P1 amplitude was decreased 2 weeks (T2) after surgery; it returned to baseline 6 weeks (T6) after surgery. FPs, FAs, and histology showed partial repopulation of Bruch's membrane by hypopigmented RPE cells and atrophied outer segments at T2. At T6, normally pigmented RPE cells were identified, and the photoreceptor layer was restored. CONCLUSION: This is the first study to show that the histological regeneration of hypopigmented RPE correlates to a return of the retinal function, measured by mfERG.

Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care.

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.

Determination of Micropulse Modes with Targeted Damage to the Retinal Pigment Epithelium Using Computer Modeling for the Development of Selective Individual Micropulse Retinal Therapy.

PURPOSE: When using a serial laser system for selective impact on the retinal pigment epithelium (RPE), there is a challenge to determine the optimal range of micropulse parameters which result in targeted damage to the RPE. This study proposes a computer model that has identified the optimal parameters to be applied. METHODS: This study was conducted on 18 patients who were diagnosed with acute central serous chorioretinopathy and transparent optical media, aged 35 to 46 years old, and type 2 and 3 on the Fitzpatrick scale. Testing of the micropulse mode was performed on the Navilas 577s laser system; 864 spots were analyzed in total. Considering the probability of damage visualization at different laser power, the computer simulation of tissue heating and protein denaturation was performed to determine the micropulse modes which resulted in selective damage to the RPE. RESULTS: The computer model parameter ΔE = 3.34 × 105 J/mol was determined from fitting the model predictions to the autofluorescence test results. The micropulse modes with a micropulse duration of 50-100 µs, duty cycle 2.4-4.8%, 10 ms-pulse envelope (5 micropulses), and spot diameter of 100 µm have efficiency and selectivity above 67% and correspond to the optimal therapeutic window for targeted RPE damage at a certain power. Increasing the micropulse duration, number of micropulses, and duty cycle leads to a decrease in the selective effect on the RPE and higher damage to adjacent tissues. CONCLUSION: The concepts of efficiency and selectivity have been introduced to quantify the amount of damage caused. The optimal range of micropulse parameters which result in effective and selective damage on the RPE has been determined for the Navilas 577s laser system. The proposed method can be used for any other serial laser system. A comparison of the different micropulse modes, as well as the CW modes, has been performed.

Cell proliferation in human epiretinal membranes: characterization of cell types and correlation with disease condition and duration.

PURPOSE: To quantify the extent of cellular proliferation and immunohistochemically characterize the proliferating cell types in epiretinal membranes (ERMS) from four different conditions: proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy, post-retinal detachment, and idiopathic ERM. METHODS: Forty-six ERMs were removed from patients undergoing vitrectomy and immediately fixed in paraformaldehyde. The membranes were processed whole and immunolabeled with either anti-MIB-1 or anti-SP6 to detect the K(i)-67 protein in proliferating cells, in combination with anti-glial fibrillary acidic protein or anti-vimentin to identify glia, anti-ezrin to identify retinal pigment epithelial cells, Ricinus communis to identify immune cells, and Hoechst to label nuclei. Digital images were collected using a laser scanning confocal microscope. The cell types were identified, their combined proliferative indices were tabulated as the average number of anti-K(i)-67-positive cells/mm(2) of tissue, and the number of dividing cells was related to the specific ocular condition and estimated disease duration. RESULTS: ERMs of all four types were shown to be highly cellular and contained proliferating cells identified as glia, retinal pigment epithelium, and of immune origin. In general, membranes identified as PVR had many more K(i)-67-positive cells in comparison to those in the other three categories, with the average number of K(i)-67-positive cells identified per mm(2) of tissue being 20.9 for proliferative diabetic retinopathy, 138.3 for PVR, 12.2 for post-retinal detachment, and 19.3 for idiopathic ERM. While all membrane types had dividing cells, their number was a relatively small fraction of the total number of cells present. CONCLUSIONS: The four ERM types studied demonstrated different cell types actively dividing at the time of removal, confirming that proliferation is a common event and does continue over many months. The low number of dividing cells at the time of removal in comparison to the total number of cells present, however, is an indicator that proliferation alone may not be responsible for the problems observed with the ERMs. Treatment strategies may need to take into consideration the timing of drug administration, as well as the contractile and possibly the inflammatory characteristics of the membranes to prevent the ensuing effects on the retina.

Selective retinal therapy with microsecond exposures using a continuous line scanning laser.

PURPOSE: To evaluate the safety, selectivity, and healing of retinal lesions created using a continuous line scanning laser. METHODS: A 532-nm Nd:YAG laser (PASCAL) with retinal beam diameters of 40 µm and 66 µm was applied to 60 eyes of 30 Dutch-belted rabbits. Retinal exposure duration varied from 15 µs to 60 µs. Lesions were acutely assessed by ophthalmoscopy and fluorescein angiography. Retinal pigment epithelial (RPE) flatmounts were evaluated with live-dead fluorescent assay. Histological analysis was performed at 7 time points from 1 hour to 2 months. RESULTS: The ratios of the threshold of rupture and of ophthalmoscopic visibility to fluorescein angiography visibility (measures of safety and selectivity) increased with decreasing duration and beam diameter. Fluorescein angiography and live-dead fluorescent assay yielded similar thresholds of RPE damage. Above the ophthalmoscopic visibility threshold, histology showed focal RPE damage and photoreceptor loss at 1 day, without inner retinal effects. By 1 week, photoreceptor and RPE continuity was restored. By 1 month, photoreceptors appeared normal. CONCLUSION: : Retinal therapy with a fast scanning continuous laser achieves selective targeting of the RPE and, at higher power, of the photoreceptors without permanent scarring or inner retinal damage. Continuous scanning laser can treat large retinal areas within standard eye fixation time.

A novel quantitative analysis method for idiopathic epiretinal membrane.

PURPOSE: To introduce a novel method to quantitively analyse in three dimensions traction forces in a vast area of the ocular posterior pole. METHODS: Retrospective analysis of 14 eyes who underwent peeling surgery for idiopathic, symptomatic and progressive epiretinal membrane. The technique measures the shift in position of vascular crossings after surgery from a fixed point, which is the retinal pigmented epithelium. This shift is defined as the relaxation index (RI) and represents a measure of the postoperative movement of the retina due to released traction after surgery. RESULTS: Best-corrected visual acuity was significantly better than baseline at all follow ups while the RI had its maximum value at baseline. Moreover, we found a significant correlation between best-corrected visual acuity at 6 months and RI at baseline. CONCLUSION: While all previous published methods focused on bi-dimensional changes observed in a small region, this study introduces a three-dimensional assessment of tractional forces. Future integration of RI into built-in processing software will allow systematic three-dimensional measurement of intraretinal traction.

Changes in entropy on polarized-sensitive optical coherence tomography images after therapeutic subthreshold micropulse laser for diabetic macular edema: A pilot study.

PURPOSE: To investigate the dynamics of the healing process after therapeutic subthreshold micropulse laser (SMPL) for diabetic macular edema (DME) using polarization-sensitive optical coherence tomography (PS-OCT). METHODS: Patients with treatment-native or previously-treated DME were prospectively imaged using PS-OCT at baseline, 1, 2, 3, and 6 months. The following outcomes were evaluated: changes in the entropy value per unit area (pixel2) in the retinal pigment epithelium (RPE) on the B-scan image; changes in the entropy value in each stratified layer (retina, RPE, choroid) based on the ETDRS grid circle overlaid with en face entropy mapping, not only the whole ETDRS grid area but also a sector irradiated by the SMPL; and the relationship between edema reduction and entropy changes. RESULTS: A total of 11 eyes of 11 consecutive DME patients were enrolled. No visible signs of SMPL treatment were detected on PS-OCT images. The entropy value per unit area (pixel2) in the RPE tended to decrease at 3 and 6 months from baseline (35.8 ± 17.0 vs 26.1 ± 9.8, P = 0.14; vs 28.2 ± 18.3, P = 0.14). Based on the en face entropy mapping, the overall entropy value did not change in each layer in the whole ETDRS grid; however, decrease of entropy in the RPE was observed at 2, 3, and 6 months post-treatment within the SMPL-irradiated sectors (P < 0.01, each). There was a positive correlation between the change rate of retinal thickness and that of entropy in the RPE within the SMPL-irradiated sector at 6 months (r2 = 0.19, P = 0.039). CONCLUSION: Entropy measured using PS-OCT may be a new parameter that facilitates objective monitoring of SMPL-induced functional changes in the RPE that could not previously be assessed directly. This may contribute to a more promising therapeutic evaluation of DME. CLINICAL TRIAL: This clinical study was registered in UMIN-CTR (ID: UMIN000042420).

Novel therapy for transthyretin-related ocular amyloidosis: a pilot study of retinal laser photocoagulation.

OBJECTIVE: The occurrence of ocular complications associated with transthyretin-related familial amyloidotic polyneuropathy increases with time, even after liver transplantation, which leads to a halt in the progression of systemic neurologic complications. This study investigated a new strategy to prevent ocular involvements. DESIGN: Two interventional case reports. PARTICIPANTS: Two patients with familial amyloidotic polyneuropathy who had progressive ocular manifestations. METHODS: We used panretinal laser photocoagulation, which damages the retinal pigment epithelium, the main location for synthesis of amyloidogenic transthyretin in ocular tissues, to treat 1 eye of each patient. After laser photocoagulation, we performed general ophthalmic examinations every 3 months for 3 years. MAIN OUTCOME MEASURES: Fundus photography, visual acuity, and intraocular pressure. RESULTS: Panretinal laser photocoagulation clearly prevented progression of amyloid deposition in the vitreous and on the retinal surface in both cases during 3 years of follow-up. No serious complications occurred. CONCLUSIONS: Panretinal laser photocoagulation is a safe and well-known procedure that offers a new treatment option to mitigate ocular manifestations in patients with transthyretin-related familial amyloidotic polyneuropathy.

Change of morphological and functional characteristics of retinal pigment epithelium cells during cultivation of retinal pigment epithelium-choroid perfusion tissue culture.

AIMS: To evaluate the changes of morphological and functional characteristics of the retinal pigment epithelium (RPE)-choroid perfusion culture during cultivation. METHODS: PorcineRPE-choroid tissue was cultivated in a perfusion tissue culture system. After the indicated times, histology, immunolocalization of collagen IV and von Willebrand factor, RPE cell viability with calcein-AM, TUNEL assay and occludin immunolocalization of RPE cells were examined. The tissue was treated with selective RPE treatment laser after different time periods and the wound healing response was characterized. Vascular endothelial growth factor secretion was measured by enzyme-linked immunosorbent assay. RESULTS: On day 8, prominent morphological degenerative changes of RPE cells were observed in histology. According to the immunohistochemistry for collagen IV, the Bruch's membrane did not display any obvious decomposition until day 8. Von Willebrand factor staining decreased during cultivation, especially at the choriocapillaris. Calcein-AM staining and TUNEL assay displayed the increase of apoptotic changes in only a minority of the cells on day 4, but in many cells on day 8. Occludin delocalization was observed on day 8. Selective RPE treatment laser-produced wounds were completely closed by monolayer RPE when wounded on fresh and 3-day-old cultures, but not when wounded on 6-day-old cultures. Vascular endothelial growth factor secretion was stable between days 2 and 5, but increased after that. CONCLUSION: Under the stated culture perfusion conditions, porcine RPE-choroid tissue was suitable for experimentation up to 5 days of maintenance.

[Atrophy of retinal pigment epithelium after macular hole surgery]. / Atrofia epiteliului pigmentar dupa chirurgia gaurii maculare.

We present three cases of macular hole. After macular surgery we observed good anatomical results (closed macular holes) but poor functional results. We try to identified the element who produced atrophy of retinal pigment epithelium.

Selective retina therapy monitoring by speckle variance optical coherence tomography for dosimetry control.

SIGNIFICANCE: Selective retina therapy (SRT) selectively targets the retinal pigment epithelium (RPE) and reduces negative side effects by avoiding thermal damages of the adjacent photoreceptors, the neural retina, and the choroid. However, the selection of proper laser energy for the SRT is challenging because of ophthalmoscopically invisible lesions in the RPE and different melanin concentrations among patients or even regions within an eye. AIM: We propose and demonstrate SRT monitoring based on speckle variance optical coherence tomography (svOCT) for dosimetry control. APPROACH: M-scans, time-resolved sequence of A-scans, of ex vivo bovine retina irradiated by 1.7-µs duration laser pulses were obtained by a swept-source OCT. SvOCT images were calculated as interframe intensity variance of the sequence. Spatial and temporal temperature distributions in the retina were numerically calculated in a 2-D retinal model using COMSOL Multiphysics. Microscopic images of treated spots were obtained before and after removing the upper neural retinal layer to assess the damage in both RPE and neural layers. RESULTS: SvOCT images show abrupt speckle variance changes when the retina is irradiated by laser pulses. The svOCT intensities averaged in RPE and photoreceptor layers along the axial direction show sharp peaks corresponding to each laser pulse, and the peak values were proportional to the laser pulse energy. The calculated temperatures in the neural retina layer and RPE were linearly fitted to the svOCT peak values, and the temperature of each lesion was estimated based on the fitting. The estimated temperatures matched well with previously reported results. CONCLUSION: We found a reliable correlation between the svOCT peak values and the degree of retinal lesion formation, which can be used for selecting proper laser energy during SRT.

Influence of age on retinochoroidal healing processes after argon photocoagulation in C57bl/6j mice.

PURPOSE: To analyze the influence of age on retinochoroidal wound healing processes and on glial growth factor and cytokine mRNA expression profiles observed after argon laser photocoagulation. METHODS: A cellular and morphometric study was performed that used 44 C57Bl/6J mice: 4-week-old mice (group I, n=8), 6-week-old mice (group II, n=8), 10-12-week-old mice (group III, n=14), and 1-year-old mice (group IV, n=14). All mice in these groups underwent a standard argon laser photocoagulation (50 microm, 400 mW, 0.05 s). Two separated lesions were created in each retina using a slit lamp delivery system. At 1, 3, 7, 14, 60 days, and 4 months after photocoagulation, mice from each of the four groups were sacrificed by carbon dioxide inhalation. Groups III and IV were also studied at 6, 7, and 8 months after photocoagulation. At each time point the enucleated eyes were either mounted in Tissue Tek (OCT), snap frozen and processed for immunohistochemistry or either flat mounted (left eyes of groups III and IV). To determine, by RT-PCR, the time course of glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and monocyte chemotactic protein-1 (MCP-1) gene expression, we delivered ten laser burns (50 microm, 400 mW, 0.05 s) to each retina in 10-12-week-old mice (group III', n=10) and 1-year-old mice (group IV', n=10). Animals from Groups III' and IV' had the same age than those from Groups III and IV, but they received ten laser impacts in each eye and served for the molecular analysis. Mice from Groups III and IV received only two laser impacts per eye and served for the cellular and morphologic study. Retinal and choroidal tissues from these treated mice were collected at 16 h, and 1, 2, 3, and 7 days after photocoagulation. Two mice of each group did not receive photocoagulation and were used as controls. RESULTS: In the cellular and morphologic study, the resultant retinal pigment epithelium interruption expanse was significantly different between the four groups. It was more concise and smaller in the oldest group IV (112.1 microm+/-11.4 versus 219.1 microm+/-12.2 in group III) p<0.0001 between groups III and IV. By contrast, while choroidal neovascularization (CNV) was mild and not readily identifiable in group I, at all time points studied, CNV was more prominent in the (1-year-old mice) Group IV than in the other groups. For instance, up to 14 days after photocoagulation, CNV reaction was statistically larger in group IV than in group III ((p=0.0049 between groups III and IV on slide sections and p<0.0001 between the same groups on flat mounts). Moreover, four months after photocoagulation, the CNV area (on slide sections) was 1,282 microm(2)+/-90 for group III and 2,999 microm(2)+/-115 for group IV (p<0.0001 between groups III and IV). Accordingly, GFAP, VEGF, and MCP-1 mRNA expression profiles, determined by RT-PCR at 16 h, 1, 2, 3, and 7 days postphotocoagulation, were modified with aging. In 1-year-old mice (group IV), GFAP mRNA expression was already significantly higher than in the younger (10-12 week) group III before photocoagulation. After laser burns, GFAP mRNA expression peaked at 16-24 h and on day 7, decreasing thereafter. VEGF mRNA expression was markedly increased after photocoagulation in old mice eyes, reaching 2.7 times its basal level at day 3, while it was only slightly increased in young mice (1.3 times its level in untreated young mice 3 days postphotocoagulation). At all time points after photocoagulation, MCP-1 mRNA expression was elevated in old mice, reaching high levels of expression at 16 h and day 3 respectively. CONCLUSIONS: Our results were based on the study of four different age groups and included not only data from morphological observations but also from a molecular analysis of the various alterations of cytokine signaling and expression. One-year-old mice demonstrated more extensive CNV formation and a slower pace of regression after laser photocoagulation than younger mice. These were accompanied by differences in growth factors and cytokine expression profiles indicate that aging is a factor that aggravates CNV. The above results may provide some insight into possible therapeutic strategies in the future.

Surgical outcomes of epiretinal membranes associated with combined hamartoma of the retina and retinal pigment epithelium.

PURPOSE: To determine the surgical outcomes of epiretinal membranes associated with combined hamartoma of the retina and retinal pigment epithelium after pars plana vitrectomy and membrane peeling with or without assistance of autologous plasmin enzyme. METHODS: Retrospective review of 11 pediatric eyes that underwent pars plana vitrectomy with membrane peeling with or without autologous plasmin enzyme. Preoperative and postoperative assessments of visual function and retinal architecture were performed by indirect ophthalmoscopy, optical coherence tomography imaging, fundus photography, and measurement of visual acuity. RESULTS: The mean age of the patients was 4.6 years (range, 1-14). Mean follow-up was 15.6 months (range, 6-42 months). The lesions were located solely in the macula in 8 of 11 (73%) patients and in the macula and posterior pole in 3 of 11 (27%) patients. Of the 11 eyes, 6 were preoperatively injected with autologous plasmin enzyme to assist in removal of the posterior hyaloid. All 11 patients (100%) had complete macular reattachment postoperatively. Eight of 11 (73%) showed improved visual acuity postoperatively, and 3 of 11 showed stabilized vision. Eight eyes required only one surgery. Four eyes (36.6%) had recurrences of epiretinal membrane, and three of these eyes required additional surgery. Of the eyes with preoperative plasmin injection, 4 of 6 (66%) showed an improvement in visual acuity whereas 2 of 6 (33%) showed stabilization of visual acuity. Four of five without plasmin showed visual improvement, and one of five had stabilization of vision. CONCLUSION: In the pediatric population, pars plana vitrectomy with membrane peeling with or without the use of autologous plasmin enzyme for epiretinal membrane associated with combined hamartomas of the retina and retinal pigment epithelium can result in improved retinal architecture and visual acuity. Visual acuity may improve despite recurrence of the epiretinal membrane.