Zachary L. Lukowski

Ab-Externo AAV-Mediated Gene Delivery to the Suprachoroidal Space Using a 250 Micron Flexible Microcatheter

Background The current method of delivering gene replacement to the posterior segment of the eye involves a three-port pars plana vitrectomy followed by injection of the agent through a 37-gauge cannula, which is potentially wrought with retinal complications. In this paper we investigate the safety and efficacy of delivering adeno-associated viral (AAV) vector to the suprachoroidal space using an ab externo approach that utilizes an illuminated microcatheter. Methods 6 New Zealand White rabbits and 2 Dutch Belted rabbits were used to evaluate the ab externo delivery method. sc-AAV5-smCBA-hGFP vector was delivered into the suprachoroidal space using an illuminated iTrackTM 250A microcatheter. Six weeks after surgery, the rabbits were sacrificed and their eyes evaluated for AAV transfection using immunofluorescent antibody staining of GFP. Results Immunostaining of sectioned and whole-mounted eyes demonstrated robust transfection in all treated eyes, with no fluorescence in untreated control eyes. Transfection occurred diffusely and involved both the choroid and the retina. No apparent adverse effects caused by either the viral vector or the procedure itself could be seen either clinically or histologically. Conclusions The ab externo method of delivery using a microcatheter was successful in safely and effectively delivering a gene therapy agent to the suprachoroidal space. This method presents a less invasive alternative to the current method of virally vectored gene delivery.

Prevention of ocular scarring post glaucoma filtration surgery using the inflammatory cell and platelet binding modulator saratin in a rabbit model.

Clinical Relevance Late complications can occur with use of current antimetabolites to prevent scarring following glaucoma filtration surgery (GFS). Safer, more targeted, anti-fibrosis agents are sought. Objectives The protein saratin has been shown to exhibit anti-fibrotic and anti-thrombotic properties in response to injury, but had not been used for glaucoma surgery. The goal of this study was to compare the efficacy of saratin with that of the widely accepted mitomycin-C (MMC) in prolonging bleb survival following GFS in the rabbit model. Two saratin delivery routes were compared; a single intraoperative topical application versus a combination of intraoperative topical application with two additional postoperative injections. Methods Twenty-four New Zealand White rabbits underwent GFS and received either intraoperative topical saratin, intraoperative topical saratin plus two injections on post-operative days 4 and 8, balanced saline solution (BSS), or MMC. The bleb tissues and their elevation durations were compared based on clinical and histological findings. Results Rabbits receiving topical+injections of saratin had a mean bleb survival of 33.6±8.5 days, significantly higher than the negative BSS controls, which averaged 17.4±6.0 days (p = 0.018). No improvement over BSS was seen for rabbits receiving topical saratin only (15.5±4.8 days, p = 0.749). Rabbits receiving saratin did not develop bleb avascularity and thinning associated with MMC treatment and there were no apparent clinical signs of toxicity. Conclusions Treatment with a single intraoperative topical application plus two additional postoperative injections significantly prolonged bleb elevation comparable to MMC, but without toxicity; however, topical application alone was ineffective.

Prevention of ocular scarring after glaucoma filtering surgery using the monoclonal antibody LT1009 (Sonepcizumab) in a rabbit model.

Purpose:Excessive scarring leading to failure of the filtering bleb continues to be a major problem after glaucoma filtration surgery. This study examines the antifibrotic effects of the anti-S1P monoclonal antibody LT1009 (Sonepcizumab) in prolonging bleb survival in a rabbit model of glaucoma filtering surgery. Methods:The frequency of LT1009 dosage was determined initially using an enzyme-linked immunosorbent assay assay measuring LT1009 eye tissue retention in 6 New Zealand White rabbits. A further 21 New Zealand White rabbits underwent glaucoma filtering surgery. Bleb tissues were observed and compared clinically and histologically. The duration of bleb elevation was compared among LT1009, balanced saline solution (BSS) negative control, and mitomycin-C (MMC)-positive control. Results:The mean duration of bleb survival was 28.5±8.5 days for rabbits receiving injections of LT1009, 21.0±5.6 days for those receiving injections of BSS, and 33.8±5.6 days for rabbits receiving MMC. Analysis of variance with post hoc testing suggests a statistically significant trend of improvement in bleb duration for LT1009 when compared with BSS controls. Nonpainful, upper eyelid edema was noted after 5 injections of LT1009, which resolved over a 10-day period. MMC eyes developed avascular conjunctivas with areas of thinning and sparse cellularity, whereas the conjunctiva of LT1009 and BSS eyes remained relatively normal. Conclusions:The monoclonal antibody LT1009 demonstrated a longer duration of bleb elevation than BSS control without adverse conjunctival effects associated with MMC. However, after multiple doses LT1009 use was associated with short-term upper eyelid edema.

Sequential Therapy with Saratin, Bevacizumab and Ilomastat to Prolong Bleb Function following Glaucoma Filtration Surgery in a Rabbit Model.

To determine if sequential treatment with Bevacizumab (Avastin), a monoclonal, VEGF antibody that blocks angiogenesis; Saratin, a 12 kD polypeptide with anti-inflammatory and anti-thrombotic properties; and Ilomastat, a matrix metalloproteinase (MMP) inhibitor, prolongs bleb life following glaucoma filtration surgery (GFS) in a rabbit model. Thirty-two New Zealand White rabbits (eight rabbits per group) underwent GFS in the left eye. Group 1 received a perioperative injection of both Saratin and Bevacizumab, and later, subconjuctival injections of Ilomastat on days 8 and 15. Group 2 received only Saratin perioperatively, and also received Ilomastat injections on days 8 and 15. Group 3, the negative control, received a single perioperative injection of Balanced Saline Solution (BSS) along with post-operative BSS injections on days 8 and 15. Group 4, the positive control, received topical treatment with Mitomycin-C (MMC) at the time of surgery with no further treatment. Blebs were evaluated by an observer masked to treatment every third day. Histology was obtained on two eyes in each group on post-op day twelve as well as all eyes following bleb failure. Eyes in group 1 had a mean bleb survival time of 29 ± 2.7 days, whereas those in group 2 that received the experimental treatment without Bevacizumab had a mean survival time of 25.5 ± 2.7 days. An ANOVA test showed that the Saratin/Ilomastat/Bevacizumab group demonstrated a significant prolongation of bleb survival compared to the BSS control—mean survival time of 19.7 ±2.7 days—(p = 0.0252) and was not significantly different from the MMC positive control group (p = 0.4238)—mean survival time of 32.5 ± 3.3. From tissue histology at day 12, the four different groups showed marked differences in the cellularity and capsule fibrosis. The MMC eyes showed minimal cellularity, were avascular and had minimal fibrous tissue. BSS group showed high cellularity, moderate to high fibrosis, and thicker and more defined capsules than either of the treatment groups and the positive control. Both the Saratin/Ilomastat/Bevacizumab and Saratin/Ilomastat only eyes showed moderate cellularity with minimal fibrosis, with less cellularity and fibrosis present in the triple treatment group. Sequential therapy with multiple agents, including Bevacizumab, prolonged bleb function following GFS in the rabbit model and were significantly better than the negative BSS control. The experimental group did not show the same surface tissue histological thinning and side effects associated with MMC treatment.

Comparison of Single versus Multiple Injections of the Protein Saratin for Prolonging Bleb Survival in a Rabbit Model

PURPOSE We compared the anti-fibrotic effects of single versus multiple postoperative injections of saratin following glaucoma filtration surgery (GFS) in the rabbit model. METHODS The experiment was in two parts. To determine the optimal frequency for postoperative therapy, seven New Zealand White (NZW) rabbits received an injection of saratin under the superior conjunctiva bilaterally, and ocular tissue concentration was determined using Western blot and bicinchoninic acid (BCA) assay. Next, 32 additional NZW rabbits underwent filtration surgery and received either single or multiple-dose saratin treatments. Mitomycin-C (MMC) and balanced saline solution (BSS) treatment acted as positive and negative controls, respectively. RESULTS Rabbits receiving only one perioperative saratin injection had a mean bleb survival time of 29.8 ± 5.3 days, while those receiving multiple (either 3 or 5+) injections of saratin had mean bleb survival times of 26.3 ± 8.1 and 26.4 ± 4.2 days, respectively. Analysis of variance with post-hoc testing showed the single injection group had a statistically favorable effect on bleb survival duration compared to BSS controls and was not significantly different from MMC. The conjunctivas of the saratin-treated rabbits did not show the thinning or avascularity that was seen in the MMC treatment group. Rabbits receiving more than three injections of saratin suffered temporary conjunctival redness and two rabbits had upper eyelid edema. CONCLUSIONS A single postoperative injection of saratin was able to prolong the duration of bleb elevation when compared to BSS controls. Additional treatments of saratin seemed to reduce effectiveness and caused short-term eye inflammation.

Comparing glaucomatous disc change using stereo disc viewing and the MatchedFlicker programme in glaucoma experts and trainees

Background/aims The objective of this study is to evaluate the accuracy and speed of trainees and experienced glaucoma specialists using the MatchedFlicker software against the manual examination of stereoscopic disc photographs for detecting glaucomatous optic disc change. Methods Three experienced glaucoma specialists, two resident ophthalmologists and one glaucoma fellow from multiple institutions independently evaluated the same 140 image pairs from 100 glaucomatous/ocular hypertensive eyes using a handheld stereo viewer and the MatchedFlicker programme. Fifty had progression to glaucoma as determined by the Ocular Hypertension Treatment Study (OHTS) Optic Disc Reading Group and endpoint committee, and 50 more were negative controls for progression with photos taken a few minutes apart. Twenty photo pairs from each of the two groups were duplicated for reviewer variability analysis. The initial viewing method was randomised and then alternated for each group of 70 image pairs. Reviewer accuracy and evaluation time for each method were measured. Results Evaluators averaged 8.6 s faster per image pair (26%) with the MatchedFlicker programme than with the stereo viewer (p=0.0007). Evaluators correctly identified more image pairs when using the MatchedFlicker software over the stereo viewer (p=0.0003). There was no significant difference between the expert and trainee group in speed or overall accuracy for either method. Experts were significantly more consistent than trainees with the duplicate image pairs (p=0.029). Trainees appeared more reluctant to designate eyes as showing glaucoma progression than experts. Conclusions Both expert glaucoma specialists and ophthalmologists in various stages of training had greater accuracy and speed with the MatchedFlicker programme than with a handheld stereoscopic viewer.

The impact of conjunctival flap method and drainage cannula diameter on bleb survival in the rabbit model

Purpose To examine the effect of cannula diameter and conjunctival flap method on bleb survival in rabbits undergoing cannula-based glaucoma filtration surgery (GFS). Methods Twelve New Zealand White rabbits underwent GFS in both eyes. The twenty-four eyes were divided into four groups. Two of the four groups (N = 12) received limbus-based conjunctival flaps (LBCF), and the other two (N = 12) received fornix-based conjunctival flaps (FBCF). Six FBCF rabbit eyes were implanted with 22-gauge drainage tubes, and the other six were implanted with 26-gauge tubes. Likewise, six LBCF rabbits received 22-gauge drainage tubes and six received 26-gauge tubes. Filtration blebs were evaluated every three days by a masked observer. Bleb failure was defined as the primary endpoint in this study and was recorded after two consecutive flat bleb evaluations. Results Group 1 (LBCF, 22- gauge cannula) had a mean bleb survival time (Mean ± SD) of 18.7 ± 2.9 days. Group 2 (LBCF, 26-gauge cannula) also had a mean bleb survival time of 18.7 ± 2.9 days. Group 3 (FBCF, 22-gauge cannula) had a mean bleb survival time of 19.2 ± 3.8 days. Group 4 (FBCF, 26-gauge cannula) had a mean bleb survival time of 19.7 ± 4.1 days. A 2-way analysis of variance showed that neither surgical approach nor cannula gauge made a statistically significant difference in bleb survival time (P = 0.634 and P = 0.874). Additionally, there was no significant interaction between cannula gauge and conjunctival flap approach (P = 0.874), suggesting that there was not a combination of drainage gauge and conjunctival flap method that produced superior bleb survival. Conclusion Limbus and fornix-based conjunctival flaps are equally effective in promoting bleb survival using both 22 and 26-gauge cannulas in the rabbit model. The 26-gauge drainage tube may be preferred because its smaller size facilitates the implantation process, reducing the risk of corneal contact.

Intralenticular Ozurdex® – One Year Later

Reported here is a case of intralenticular sustained-release dexamethasone implant (Ozurdex®, Allergan, Irvine, CA, USA) present for 1 year with effective treatment of refractory diabetic macular edema without rapid cataract formation. The crystalline lens remained stable for 12 months on exam despite the presence of the steroid-secreting foreign body. The diabetic macular edema resolved on exam and on optical coherence tomography. After 1 year, cataract extraction was uneventfully performed by phacoemulsification for a mild decline in visual acuity. Macular edema remains resolved 2 months following cataract removal. This is the longest reported period of observation of intralenticular Ozurdex in the literature. Ozurdex remains effective despite intralenticular location, and it can have minimal effects on cataract progression.