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Retina  |   October 2012
Evaluation of Ultrasound-Assisted Thrombolysis Using Custom Liposomes in a Model of Retinal Vein Occlusion
Author Affiliations & Notes
  • Walid F. Abdallah
    From the Doheny Eye Institute, Los Angeles, California; the
    Department of Ophthalmology, Faculty of Medicine, Zagazig University, Zagazig, Egypt; the departments of
  • Hitenkumar Patel
    Radiology and
  • Edward G. Grant
    Radiology and
  • Bruno Diniz
    From the Doheny Eye Institute, Los Angeles, California; the
    Department of Ophthalmology - Universidade Federal de São Paulo, (UNIFESP/EPM), São Paulo, Brazil; and the
  • Gerald J. Chader
    From the Doheny Eye Institute, Los Angeles, California; the
  • Mark S. Humayun
    From the Doheny Eye Institute, Los Angeles, California; the
    Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, California; the
    Department of Biomedical Engineering, University of Southern California, Los Angeles, California.
  • Corresponding author: Mark S. Humayun, Professor of Ophthalmology, Biomedical Engineering, Cell & Neurobiology, Doheny Eye Institute, 1355 San Pablo Street, Los Angeles, CA 90033; Humayun@usc.edu
Investigative Ophthalmology & Visual Science October 2012, Vol.53, 6920-6927. doi:10.1167/iovs.12-10389
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      Walid F. Abdallah, Hitenkumar Patel, Edward G. Grant, Bruno Diniz, Gerald J. Chader, Mark S. Humayun; Evaluation of Ultrasound-Assisted Thrombolysis Using Custom Liposomes in a Model of Retinal Vein Occlusion. Invest. Ophthalmol. Vis. Sci. 2012;53(11):6920-6927. doi: 10.1167/iovs.12-10389.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To study the potential efficacy of ultrasound (US) assisted by custom liposome (CLP) destruction as an innovative thrombolytic tool for the treatment of retinal vein occlusion (RVO).

Methods.: Experimental RVO was induced in the right eyes of 40 rabbits using laser photothrombosis; the US experiment took place 48 hours later. Rabbits were randomly divided into four equal groups: US+CLP group, US+saline group, CLP+sham US group, and no treatment group. The latter three groups acted as controls. Fundus fluorescein angiography and Doppler US were used to evaluate retinal blood flow.

Results.: CLP-assisted US thrombolysis resulted in restoration of flow in seven rabbits (70%). None of the control groups showed significant restoration of retinal venous blood flow.

Conclusions.: US-assisted thrombolysis using liposomes resulted in a statistically significant reperfusion of retinal vessels in the rabbit experimental model of RVO. This approach might be promising in the treatment of RVO in humans. Further studies are needed to evaluate this approach in patients with RVO. Ultrasound assisted thrombolysis can be an innovative tool in management of retinal vein occlusion.

Introduction
Retinal vein occlusion (RVO) is the second leading cause of retinal vascular disease after diabetic retinopathy, 1 with a prevalence of 0.7% to 1.6%. 1,2 In a population-based study, the overall incidence of symptomatic RVO in a 4-year period was found to be 0.21% in patients aged 40 or older. 3  
The clinical characteristics, prognosis, and response to treatment are influenced by the location of the occlusion in the retinal venous vasculature and by the extent of retinal nonperfusion. 46 RVO is classified, according to where the obstruction is located, into central retinal vein (CRVO), branch retinal vein (BRVO), and hemiretinal vein occlusion (HRVO). In CRVO, the location of the occlusion may occur at the level of or posterior to the lamina cribrosa. BRVO is an occlusion of either a major branch retinal vein draining one quadrant of the retina, a macular branch vein draining a portion of the macula, or a peripheral branch vein draining a portion of the retinal periphery. 79 In eyes with a HRVO, venous outflow from the superior or inferior retina is impaired. 10 In 20% of eyes, the merger of the superior and inferior venous trunks occurs posterior to the lamina cribrosa. 11 If one of these trunks becomes occluded, an HRVO occurs. 1012  
Current treatment options for RVO include laser photocoagulation, 1315 intravitreal steroid or anti-VEGF drug injection, 1625 and less frequently, vitrectomy with and without sheathotomy. 2632 Most of these approaches do not address the thrombus: the inciting underlying pathology. Sheathotomy does surgically try to dislodge the thrombus, but the procedure is complex. Systemic or local thrombolytic therapy using tissue plasminogen activator (tPA) or urokinase once seemed to be an interesting therapeutic modality; but these therapies have been associated with serious complications. 3339  
The development of liposomes is a translational research endeavor. Its earliest impact has been in areas of biopharmaceuticals (e.g., drug delivery, drug discovery), 4044 gene therapy, 45 and imaging. 4650 Liposomes are microspheres with unique acoustic properties that make them useful as ultrasound (US) contrast agents for sonographic imaging, especially when their cores contain air or gas. 4750 Experimental studies have shown that US-accelerated thrombolysis may be further enhanced by administration of liposome microspheres. 5162 High power US has been shown to induce cavitation of liposomes and fluid motion into the thrombus. 52,53 Specifically, application of high acoustic pressure US has been shown to induce nonlinear oscillations of liposomes, leading to a continuous absorption of energy until the microspheres explode, releasing the absorbed energy. 55 The synergic effect of US and liposomes on sonothrombolysis has been demonstrated in clinical studies in patients with arteriovenous dialysis graft thrombosis. 56  
The objective of the present study was to test the innovative application of US-mediated liposome destruction in the treatment of RVO in an experimental animal model induced by laser photothrombosis (LPT), which depends on the use of rose bengal; a dye that, when exposed to light at its peak absorption wavelength (550 nm), generates oxygen singlet. This method seems to fairly well reflect the situation in the human disease. Peroxidation of endothelial cell lipids by singlet oxygen is the mechanism of membrane damage and, in blood vessels, may serve as the initial stimulus for platelet adhesion and aggregation. 6366 Animal studies proved that laser-treated retinal vessels had a platelet-rich clot associated with injured endothelium, but there is no electron microscopic evidence of fibrin or damage to cells adjacent to the endothelial cells (e.g., smooth-muscle cells in the walls of arterioles). 6769  
Materials and Methods
Animal Preparation
Forty age-matched, Dutch pigmented normal rabbits, each weighing 2 to 3 kg, were used. All animal experiments adhered to the regulations of the Institutional Animal Care and Use Committee of University of Southern California and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. For all animal procedures, the rabbits were anesthetized by intramuscular injection of a mixture of ketamine hydrochloride (25 mg/kg) and xylazine hydrochloride (6 mg/kg). The pupils were dilated with a topical application of phenylephrine hydrochloride 2.5% and tropicamide 0.5% eye drops. 
Fundus Photography
All rabbits were scheduled for baseline imaging before LPT; imaging was repeated once, 48 hours after laser, and again, immediately after the US experiment. A digital fundus camera system (FF 450Plus; Carl Zeiss Meditec AG, Jena, Germany) was used to obtain color fundus photographs and for fundus fluorescein angiography (FFA). The latter procedure was used to document the status of blood flow in retinal vessels. 
RVO Model
Laser photothrombosis of a main branch retinal vein in the right eye of each rabbit was performed as described previously. 70 Briefly, the 532-nm laser (Iris Medical Oculight Glx; IRIDEX Corporation, Mountain View, CA) was applied to the retinal veins (guided by the baseline FFA photos) at or close to the disc margin, 10 seconds after intravenous injection of a dose of 40 mg/kg rose bengal dye (Sigma-Aldrich, Inc., St. Louis, MO). Laser shots were applied to either the main nasal or temporal retinal veins or to both. Care was taken to avoid damaging the adjacent arteries. Laser parameters were as follows: spot size, 125 μm; duration, 500 ms; power starting at 150 mW and rising to 300 mW after a few shots. A total of 10 to 30 shots were applied to the treatment spot (average 20). Whitening of the treated segment of the vein, distal engorgement, and proximal blanching were considered the treatment endpoints. Complete RVO was confirmed by FFA on the day of the US experiment. Rabbits with good occlusion on at least one side (i.e., nasal or temporal) were included in the study, while those with poor or no vein occlusion were excluded from the study. 
Randomization and Animal Subgroups
Rabbits that were included in the study were divided into four equal groups: Group 1 (US+custom liposome [CLP]) received CLP-assisted US thrombolysis 48 hours after LPT. Group 2 (US+saline) received normal saline infusion during therapeutic US application. Group 3 (CLP+sham US) received CLP infusion, while the therapeutic US was turned off. Group 4 (no treatment) received 100 mL normal saline infusion, while the therapeutic US was turned off. 
To reduce confounding by independent variables that have not been accounted for in the experimental design, animals that were included in the study were assigned randomly to the four animal subgroups using computer generated randomization tables. 
CLP Preparation
The liposomes used in our study were custom built and composed of a core of sulfur hexafluoride gas and an outer shell of phospholipids (macrogol, distearoylphosphatidylcholine, dipalmitoylphosphatidylglycerol sodium, and palmitic acid). For the saline+US and the no treatment groups, 100-mL normal saline was infused intravenously (IV). 
Probe Positioning
Each rabbit was placed on its left side with the speculum in place, and US gel (Aquasonic; Parker Laboratories, Inc., Fairfield, NJ) was applied. The probe unit was then attached to a stereotactic stand, allowing proper positioning of the probe, and avoiding untoward pressure on the globe that could give rise to erroneous measurements. The probe was applied to the inferior sclera with the long axis of the probe parallel to the limbus. 
Spectral Doppler US Imaging
Measurement of the retinal vein blood velocity from both the nasal and temporal sides was obtained (as previously described), 71 both before and after therapeutic or sham US application. 
For this purpose, we used a dedicated, small animal, high resolution imaging unit (Vevo 770; Visual Sonics, Toronto, Canada) and a 20-MHz, high frequency, linear transducer (lateral resolution, 140 μm at the focal depth; axial resolution, 140 μm; focal point, 15 mm with a depth of focus of 2.7 mm). 
Therapeutic US
For therapeutic US, we applied the contrast imaging mode; we then applied 10 cycles of low-power destruction pulses (frequency, 10 MHz) every 5 minutes. For the US+saline group, 10 cycles of destruction pulses were run. For the CLP+sham US and the no treatment groups, the ultrasound probe was applied to the eye, but therapeutic US was turned off during the infusion. 
Statistical Analysis
Data are shown as mean ± SD. Comparison between means was conducted by running the one-way ANOVA test. Statistical significance was defined as P less than 0.05. All analyses were conducted using the SPSS 17.0 statistical package (SPSS Inc., Chicago, IL). 
Results
Analysis and interpretation of data obtained from FFA and Doppler flow studies were performed by an experienced co-author who was blinded to the animal subgroup when presented with the raw data. 
Fluorescein Angiography
The Doppler results were confirmed by FFA in all cases. Evaluation of the status of the retinal vein blood flow as seen by the Doppler US correlated well with the FFA findings. All animals that were included in the study had complete RVO (except four animals), which were allocated randomly to the four groups of the study, thus, each group included nine complete RVO and one partial RVO. 
Two patterns of branch retinal arterial occlusion (BRAO) developed in our study; immediate and delayed patterns. In the immediate (direct) pattern, the laser burns touched the arterial wall resulting in immediate spasm of the artery and subsequent arterial occlusion. We had four animals that developed this pattern of BRAO and these animals were excluded from the study. 
On the other hand, the delayed (indirect) pattern is the pattern that developed within 48 hours as a part of the natural course of this model of RVO particularly with the complete venous occlusion pattern. This developed in four animals and these animals were randomly allocated to the four animal subgroups. Thus, each animal subgroup included complete RVO (n = 8), complete RVO+arterial occlusion (n = 1), and partial RVO (n = 1). 
In the US+CLP group, seven rabbits (70%) showed reperfusion of the occluded retinal veins; however, the reperfused vessels were smaller in diameter than they were before the laser occlusion (Figs. 1, 2, 3). None of the control groups showed any significant restoration of blood flow in the occluded veins after the experiment. 
Figure 1. 
 
Fundus fluorescein angiographic images of a rabbit in the US+CLP group. Baseline imaging (A) shows normal retinal venous filling. Post laser imaging (B) shows no flow in a segment of the temporal retinal vein but retrograde filling from venous tributaries (indicates partial venous occlusion). Final imaging after CLP-enhanced US thrombolysis (C) shows return of flow in the occluded retinal vein and complete retinal venous filling.
Figure 1. 
 
Fundus fluorescein angiographic images of a rabbit in the US+CLP group. Baseline imaging (A) shows normal retinal venous filling. Post laser imaging (B) shows no flow in a segment of the temporal retinal vein but retrograde filling from venous tributaries (indicates partial venous occlusion). Final imaging after CLP-enhanced US thrombolysis (C) shows return of flow in the occluded retinal vein and complete retinal venous filling.
Figure 2. 
 
Fundus photography and fundus fluorescein angiogram of a rabbit in the US+CLP group with partial venous occlusion pattern. Baseline imaging shows normal retinal vessels in color photograph (A), early arterial flow shows up at 8 seconds (B), and when retinal venous filling is complete (C). Post laser imaging shows no flow in the nasal and temporal retinal veins in a small segment at the edge of the optic disc (D, E), while the remaining retinal veins fill late by collaterals (F). Black arrows point to the site of laser application. Final imaging after US experiment shows modest return of flow in the partially occluded retinal veins with complete filling by 22 seconds (G, H, I). White arrows point to filling of the previously occluded venous segment.
Figure 2. 
 
Fundus photography and fundus fluorescein angiogram of a rabbit in the US+CLP group with partial venous occlusion pattern. Baseline imaging shows normal retinal vessels in color photograph (A), early arterial flow shows up at 8 seconds (B), and when retinal venous filling is complete (C). Post laser imaging shows no flow in the nasal and temporal retinal veins in a small segment at the edge of the optic disc (D, E), while the remaining retinal veins fill late by collaterals (F). Black arrows point to the site of laser application. Final imaging after US experiment shows modest return of flow in the partially occluded retinal veins with complete filling by 22 seconds (G, H, I). White arrows point to filling of the previously occluded venous segment.
Figure 3. 
 
Fundus photographs and fundus fluorescein angiogram of a rabbit in the US+saline group. Images (A, B, C) represent those taken before laser photothrombosis, while (D, E, F) represent the images taken 48 hours after retinal vein occlusion, and (G, H, I) represent images taken at the end of therapeutic US application. Arrows in (D) and (F) point to site of laser application. Note the non filling of the proximal segment of the laser-treated retinal veins and the absence of filling after US application. The distal segments of these veins fill from collaterals.
Figure 3. 
 
Fundus photographs and fundus fluorescein angiogram of a rabbit in the US+saline group. Images (A, B, C) represent those taken before laser photothrombosis, while (D, E, F) represent the images taken 48 hours after retinal vein occlusion, and (G, H, I) represent images taken at the end of therapeutic US application. Arrows in (D) and (F) point to site of laser application. Note the non filling of the proximal segment of the laser-treated retinal veins and the absence of filling after US application. The distal segments of these veins fill from collaterals.
The Table shows the number of animals used in our study and the fate of each animal according to the pattern of vascular occlusion. 
Table. 
 
This Table Describes the Distribution of Animals according to the Pattern of Occlusion and the Fate of Each.
Table. 
 
This Table Describes the Distribution of Animals according to the Pattern of Occlusion and the Fate of Each.
Pattern of Vascular Occlusion n Fate of the Animal
Complete venous occlusion 36 Completed the study
Randomly assigned to the 4 groups of the study (with each group having 9 animals of this pattern)
Partial venous occlusion 4 Completed the study
Randomly assigned to the 4 groups of the study (with each group having 1 animal of this pattern)
Combined arterial+venous occlusion 5 Excluded from the study
None 4 Excluded from the study
Retinal Venous Blood Velocity Measurement
Averaged pulse Doppler measurements of blood flow velocity in the experimentally occluded retinal veins at the end of the experiment were compared with those taken at the start of therapeutic or sham US experiments (Figs. 4, 5). 
Figure 4. 
 
Graph shows the mean retinal venous blood velocity as measured before LPT (baseline), post laser, and after the US experiment (after treatment). Only the US+CLP group showed statistically significant improvement of blood flow at the end of the US experiment. *Refers to P < 0.05 with statistical significance
Figure 4. 
 
Graph shows the mean retinal venous blood velocity as measured before LPT (baseline), post laser, and after the US experiment (after treatment). Only the US+CLP group showed statistically significant improvement of blood flow at the end of the US experiment. *Refers to P < 0.05 with statistical significance
Figure 5. 
 
Spectral Doppler imaging of a rabbit in the US+CLP group shows no recordable retinal venous blood flow as measured from the retinal surface (A), while after CLP-enhanced US thrombolysis, retinal venous blood flow in the treated vein is restored, and the calculated average blood velocity is 1.0 cm/s (B).
Figure 5. 
 
Spectral Doppler imaging of a rabbit in the US+CLP group shows no recordable retinal venous blood flow as measured from the retinal surface (A), while after CLP-enhanced US thrombolysis, retinal venous blood flow in the treated vein is restored, and the calculated average blood velocity is 1.0 cm/s (B).
The mean venous blood velocity for the US+CLP group improved from 0.05 ± 0.1 cm/s before the experiment to 1.28 ± 0.9 cm/s at the end of the experiment, with a statistically significant difference (P value = 0.002). On the other hand, the final measurements of retinal venous blood velocity for the three control groups were not statistically different from the post laser measurements. 
Discussion
Although RVO represents a common, potentially blinding disease, most of the available treatment modalities are neither safe nor efficacious. This issue has led to a search for a safer and more effective treatment approach. A number of studies point to the possible value of US-assisted thrombolysis as an effective and potentially safe procedure, with several studies on experimental US thrombolysis in animals. 
The first approach examined the ability of high intensity US delivered via catheter to disrupt vascular thrombi without administration of plasminogen activator. The US frequency range was 19.5 to 26.5 kHz, and time of US application was 4 to 5 minutes. 59,61,7276 The second approach used low intensity US in the range of 0.75 to 2 W/cm2 to accelerate plasminogen-induced enzymatic thrombolysis. The US frequency range used was 20 kHz to 1MHz. 7784  
Since their introduction in the market, liposomes have been extensively studied for use in drug delivery and molecular imaging. 8587 One of the most important advantages of liposomes is the utilization of phospholipids that are natural components of cell membranes. Therefore, they can be eliminated from the body by simple degradation pathways without causing any toxic effect. 88 Similarly, numerous publications since 1995 demonstrate the safety of sulfur hexafluoride (SF6) with the major route of elimination through the lungs. 
Our approach in the present study was to use US-assisted destruction of liposomes to restore blood flow in cases with RVO. The time to apply the US pulse destruction cycles was governed by the appearance of a well-flowing CLP, as seen in the contrast imaging mode. We report success in 70% of cases treated with CLP-assisted US, without the use of any fibrinolytic agents. 
To our knowledge, the only study on the effectiveness of US-assisted thrombolysis in RVO in rabbits was performed by Larsson et al. 79 In their study, instead of liposomes they used streptokinase with US. Moreover, the only tool they used to document reperfusion was FFA, performed 12 hours after the experiment. This may have compromised their results, as our experience indicates that the laser model of RVO in rabbits can vary in terms of duration of occlusion. Specifically, the natural history of the vein occlusion itself may make the vein patent within hours of conducting the experiment. This reperfusion can be spontaneous and unrelated to the US experiment itself. We chose to perform our experiments 48 hours after LPT because, based on our previous studies, 70 maximum vascular occlusion occurs 2 days after LPT. The occluded vessels start recanalizing afterwards, reaching maximum revascularization several weeks later. 70  
The only way to obviate the possibility of natural opening of the vein occlusion is to perform FFA immediately after the experiment, as was done in our study. To further enforce our proof of assessment of the retinal blood flow, we measured the blood velocity in the retinal veins using pulse Doppler US during the experiment itself in the real time of the US experiment. 
Many challenges were encountered in our study. First, the LPT model in rabbits is not fully reliable; we excluded four rabbits that underwent light laser application because of incomplete RVO, as determined by FFA on the third day post laser. Second, measurement of blood velocity in tiny vessels such as the retinal veins is difficult, and a good deal of experience is needed to ensure accuracy. We were able to successfully identify these vessels with a combination of power Doppler with pulse Doppler mode. As soon as power Doppler located a superficial blood flow near the retinal surface, pulse Doppler was used to measure the blood velocity and identify the retinal veins by their characteristic waveforms. Third, artifacts are produced during the measurement of blood velocity by pulse Doppler when a high concentration of CLP is still in the circulation. We overcame this problem by avoiding any Doppler measurements while CLP were still in the blood stream. The CLP can be identified by the presence of spikes on the pulse Doppler and the blooming artifact on power Doppler. 83,84  
Our current study had several limitations, namely the relatively small sample of animals used, possible technical errors with Doppler US measurements, and BRAO that developed in some animals. Regarding the animal number, this study was designed as a feasibility study to better evaluate the use of US thrombolysis in RVO without fibrinolytic agents. But even with this limited number of cases, the results clearly demonstrate efficacy with the use of CLP-enhanced US. Also, the translation of this approach to patients with RVO, though exciting, should not be taken as a given and needs to be demonstrated. Patients have RVO due to a different pathophysiologic mechanism than the rabbit model in this study, and the duration of RVO is slightly different, usually longer. The latter results in structural changes to the thrombus and, possibly, in remodeling of the retinal vasculature, not to mention the long-term effects of retinal edema and hemorrhage. The safety of the technique seems good with minimal potential side effects. These do not seem to be directly related to the use of US+liposomes and are being summarized for future publication. 
Regarding BRAO, we did not exclude these four cases, as we believe that arterial stagnation or even occlusion from extension of the thrombus is a natural evolution for a perfectly complete venous occlusion and can be unavoidable incident in some cases as reported in previous studies. 70  
We hypothesize that a difference exists between the two patterns of arterial occlusion. In the immediate BRAO, severe arterial wall damage was found on histopathologic studies (unpublished data), which in turn would have complicated our interpretation of the results if we had used these animals. On the other hand, in the delayed BRAO, thrombus formation was detected in the animals of the (CLP+sham US) and the (US+saline) groups, which explains why we decided to keep the animals with this occlusion pattern included in our study. 
Regarding possible technical difficulties with Doppler measurements of retinal venous blood velocity, it was difficult to image such small vessels as the retinal veins in rabbits with 100% certainty. Hence, we relied mainly on FFA as the gold standard in order to document and confirm the presence or absence of the blood flow in the laser-treated retinal veins. However, we felt it would be more interesting to assess the flow by Doppler US using a combined technique of pulse Doppler and power Doppler in addition to fundus angiography. 
Contrast-enhanced therapeutic ultrasound may carry some risk to patients. First, a fraction of MB is likely to burst under diagnostic acoustic intensities because of bubble wall impurities. Under high acoustic pressures, the stabilizing shells of MB may rupture, freeing gas bubbles. SF6 is an inert, nontoxic, and safe gas. The route of SF6 elimination is via the lungs in the exhaled air with approximately 40% to 50% of the dose being eliminated within the first minute and with greater than 75% of the dose eliminated by 11 minutes. 89,90 Second, MB cavitation risks involve chemical hazards (the creation of reactive free radicals) and mechanical hazards to inner vascular wall tissues. 89 Third, vascular bruising or vascular rupture normally heals, but the potential for cavitation harm could be clinically significant for some patients. Patients with hemophilia or on aspirin or other anticlotting therapies may be affected by microvascular ruptures. 9193 Finally, the FDA ordered a black box warning on the use of perflutren-containing microbubbles in patients with acute coronary syndromes, acute myocardial infarction, and worsening or clinically unstable heart failure, severe emphysema and pulmonary emboli, or other conditions that cause pulmonary hypertension. 94,95  
Overall, however, microbubbles are very safe contrast agents with a minor adverse event rate of 0.13% (dizziness, erythematous rash, itching, nausea, and vomiting) and major adverse event rate of 0.0086% (dyspnea, bronchospasm, slight hypotension, clouding of consciousness, dorsolumbar pain, severe hypotension, and cutaneous rash). 96  
In our study, no major or minor adverse effects have been observed in any of the animal subgroups using FFA and optical coherence tomography (OCT) imaging, electroretinography, immunohistochemistry, and light and electron microscopy (unpublished data). 
Conclusions
Liposome-assisted US thrombolysis is an innovative therapeutic tool for RVO. Further studies are needed to study the short- and long-term effects of this therapy in order to try to develop it as a possible treatment for patients with RVO. 
Acknowledgments
The authors thank Laurie Dustin (Core statistician for the Doheny Eye Institute National Eye Institute Core grant) for statistical analysis of data. 
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Footnotes
 Supported in part by National Eye Institute Core Grant EY03040.
Footnotes
 Disclosure: W.F. Abdallah, None; H. Patel, None; E.G. Grant, None; B. Diniz, None; G.J. Chader, None; M.S. Humayun, P
Figure 1. 
 
Fundus fluorescein angiographic images of a rabbit in the US+CLP group. Baseline imaging (A) shows normal retinal venous filling. Post laser imaging (B) shows no flow in a segment of the temporal retinal vein but retrograde filling from venous tributaries (indicates partial venous occlusion). Final imaging after CLP-enhanced US thrombolysis (C) shows return of flow in the occluded retinal vein and complete retinal venous filling.
Figure 1. 
 
Fundus fluorescein angiographic images of a rabbit in the US+CLP group. Baseline imaging (A) shows normal retinal venous filling. Post laser imaging (B) shows no flow in a segment of the temporal retinal vein but retrograde filling from venous tributaries (indicates partial venous occlusion). Final imaging after CLP-enhanced US thrombolysis (C) shows return of flow in the occluded retinal vein and complete retinal venous filling.
Figure 2. 
 
Fundus photography and fundus fluorescein angiogram of a rabbit in the US+CLP group with partial venous occlusion pattern. Baseline imaging shows normal retinal vessels in color photograph (A), early arterial flow shows up at 8 seconds (B), and when retinal venous filling is complete (C). Post laser imaging shows no flow in the nasal and temporal retinal veins in a small segment at the edge of the optic disc (D, E), while the remaining retinal veins fill late by collaterals (F). Black arrows point to the site of laser application. Final imaging after US experiment shows modest return of flow in the partially occluded retinal veins with complete filling by 22 seconds (G, H, I). White arrows point to filling of the previously occluded venous segment.
Figure 2. 
 
Fundus photography and fundus fluorescein angiogram of a rabbit in the US+CLP group with partial venous occlusion pattern. Baseline imaging shows normal retinal vessels in color photograph (A), early arterial flow shows up at 8 seconds (B), and when retinal venous filling is complete (C). Post laser imaging shows no flow in the nasal and temporal retinal veins in a small segment at the edge of the optic disc (D, E), while the remaining retinal veins fill late by collaterals (F). Black arrows point to the site of laser application. Final imaging after US experiment shows modest return of flow in the partially occluded retinal veins with complete filling by 22 seconds (G, H, I). White arrows point to filling of the previously occluded venous segment.
Figure 3. 
 
Fundus photographs and fundus fluorescein angiogram of a rabbit in the US+saline group. Images (A, B, C) represent those taken before laser photothrombosis, while (D, E, F) represent the images taken 48 hours after retinal vein occlusion, and (G, H, I) represent images taken at the end of therapeutic US application. Arrows in (D) and (F) point to site of laser application. Note the non filling of the proximal segment of the laser-treated retinal veins and the absence of filling after US application. The distal segments of these veins fill from collaterals.
Figure 3. 
 
Fundus photographs and fundus fluorescein angiogram of a rabbit in the US+saline group. Images (A, B, C) represent those taken before laser photothrombosis, while (D, E, F) represent the images taken 48 hours after retinal vein occlusion, and (G, H, I) represent images taken at the end of therapeutic US application. Arrows in (D) and (F) point to site of laser application. Note the non filling of the proximal segment of the laser-treated retinal veins and the absence of filling after US application. The distal segments of these veins fill from collaterals.
Figure 4. 
 
Graph shows the mean retinal venous blood velocity as measured before LPT (baseline), post laser, and after the US experiment (after treatment). Only the US+CLP group showed statistically significant improvement of blood flow at the end of the US experiment. *Refers to P < 0.05 with statistical significance
Figure 4. 
 
Graph shows the mean retinal venous blood velocity as measured before LPT (baseline), post laser, and after the US experiment (after treatment). Only the US+CLP group showed statistically significant improvement of blood flow at the end of the US experiment. *Refers to P < 0.05 with statistical significance
Figure 5. 
 
Spectral Doppler imaging of a rabbit in the US+CLP group shows no recordable retinal venous blood flow as measured from the retinal surface (A), while after CLP-enhanced US thrombolysis, retinal venous blood flow in the treated vein is restored, and the calculated average blood velocity is 1.0 cm/s (B).
Figure 5. 
 
Spectral Doppler imaging of a rabbit in the US+CLP group shows no recordable retinal venous blood flow as measured from the retinal surface (A), while after CLP-enhanced US thrombolysis, retinal venous blood flow in the treated vein is restored, and the calculated average blood velocity is 1.0 cm/s (B).
Table. 
 
This Table Describes the Distribution of Animals according to the Pattern of Occlusion and the Fate of Each.
Table. 
 
This Table Describes the Distribution of Animals according to the Pattern of Occlusion and the Fate of Each.
Pattern of Vascular Occlusion n Fate of the Animal
Complete venous occlusion 36 Completed the study
Randomly assigned to the 4 groups of the study (with each group having 9 animals of this pattern)
Partial venous occlusion 4 Completed the study
Randomly assigned to the 4 groups of the study (with each group having 1 animal of this pattern)
Combined arterial+venous occlusion 5 Excluded from the study
None 4 Excluded from the study
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