Hani Levkovitch-Verbin

Intravitreal Injections of Neurotrophic Factors Secreting Mesenchymal Stem Cells Are Neuroprotective in Rat Eyes following Optic Nerve Transection

PURPOSE To evaluate the neuroprotective effect of intravitreal injections of neurotrophic factors secreting mesenchymal stem cells (NTF-SCs) on the survival of retinal ganglion cells (RGCs) in rat eyes after optic nerve transection (ONT). METHODS Rat and human bone marrow-derived mesenchymal stem cells (MSCs) were induced to secrete high levels of NTF. The neuroprotective effect from intravitreally injected untreated MSCs or NTF-SCs was compared with that from PBS injections using an ONT model in 146 rats. RGCs were labeled by applying rhodamine dextran to the orbital optic nerve or by injecting Fluorogold into the superior colliculus. Cell- and saline-treated eyes were compared 8 days after ONT. For tracking, MSCs and NTF-SCs were labeled with PKH26 and analyzed at 2 hours and at 10, 17, and 24 days using immunohistochemistry and RT-PCR. RESULTS Mean RGC survival at 8 days after transection increased significantly after intravitreal injections of human NTF-SCs (69% ± 3%) or of untreated human MSCs (66% ± 5%) versus PBS (46% ± 3%; P = 0.0005 and P = 0.03, respectively). In an additional set of experiments, human NTF-SCs versus PBS were significantly neuroprotective, but bone marrow-derived rat NTF-SCs were not (P = 0.001 and P = 0.1, respectively). Immunohistochemistry demonstrated that human-derived MSCs, human NTF-SCs, and rat-derived NTF-SCs survived at least 24 days after intravitreal injection. CONCLUSIONS Bone marrow-derived MSCs can deliver NTFs by intravitreal injection and can be neuroprotective after ONT. This approach might be further studied to deliver NTFs by autotransplantation in glaucomatous eyes.

Mechanism of retinal ganglion cells death in secondary degeneration of the optic nerve.

In central nervous system injury, the secondary degeneration process is known to play a major role in determining the final extent of impairment. Here, we investigated the mechanism of retinal ganglion cell (RGC) death in secondary degeneration of the optic nerve using a unique model that allows morphological separation between primary and secondary degeneration. A partial transection model was applied unilaterally in 110 Wistar rat eyes. The rate of apoptosis was evaluated in primary and secondary degeneration over a period of 6 months using the Hoechst staining technique. The involvement of caspase 3 and members of the Bcl-2 family (Bax, Bad, Bcl-2 and Bcl-xl) was evaluated at multiple time points for 6 months after the injury by immunohistochemistry and RT-PCR. We found that in secondary degeneration of the optic nerve, RGCs died by apoptosis from day 3-6 months following the injury, peaking at 3 months (16.3% +/- 2.5% apoptotic cells, p < 0.01). Both primary and secondary degeneration of the optic nerve resulted in caspase 3 activation, which was longer and more intense in the former. Similarly, both primary and secondary degeneration led to significant (p < 0.05) downregulation of the pro-survival genes Bcl-2 and Bcl-x-L and up-regulation of the pro-apoptotic genes Bax and Bad (p < 0.05), with a suggested delay in secondary degeneration. Thus, secondary degeneration of the optic nerve leads to RGC apoptosis over long periods in a similar mechanism as in primary degeneration.

Reduced Mesopic and Photopic Foveal Contrast Sensitivity in Glaucoma

OBJECTIVE To demonstrate differences in foveal contrast sensitivity (CS) between glaucomatous and nonglaucomatous eyes using a simple, rapid computerized test. METHODS This study included consecutive patients with glaucoma (35 eyes) and age-matched control participants (23 eyes) with visual acuity of 20/30 or better. Patients with any other ocular disease, including cataract, were excluded. All participants underwent a comprehensive ocular examination, perimetry, and CS. Contrast sensitivity was examined by means of 2 computerized psychophysical tests. The transient method included the presentation of a target in a temporal, 2-alternative, forced-choice procedure, and the static method involved 4 forced-choice procedures. The targets were Gabor patches with spatial frequencies of 1.5 to 9.0 cycles per degree. The tests were conducted under photopic and mesopic conditions. RESULTS Significantly lower foveal CS was found in glaucomatous eyes under photopic and mesopic conditions for all spatial frequencies (P < .01). The transient and static methods yielded similar results and were significantly correlated (P < .001). All transient photopic and mesopic CSs were significantly correlated with cup to disc ratio (P < .05). The static photopic spatial frequency of 6 cycles per degree was significantly correlated with the severity of the glaucomatous damage. CONCLUSIONS The results indicate that foveal CS is impaired in glaucoma despite good visual acuity, suggesting that central visual function damage occurs in glaucoma. The similarity between the 2 methods of testing implies that the static method, being the shorter and easier one, may be used in future research. Further research is necessary to establish a CS testing role in the screening and monitoring of glaucoma.

Constitutive Expression of HIF‐1α and HIF‐2α in Bone Marrow Stromal Cells Differentially Promotes Their Proangiogenic Properties

Bone marrow stromal cells (BMSCs) contain progenitors capable of participating in postnatal angiogenesis. Hypoxia‐inducible factors (HIFs) mediate endothelial activation by driving the expression of multiple angiogenic factors. We explored the potential of HIF‐1α and HIF‐2α modification in BMSCs, as a tool to improve cell‐based angiogenic therapy. BMSCs were retrovirally transduced to express stable forms of HIF‐1α and HIF‐2α. HIF‐1α and, to a greater extent, HIF‐2α overexpression promoted differentiation of BMSCs to the endothelial lineage, evident by CD31 and Tie‐2 expression and improved adhesive properties. Whereas chemotaxis toward stromal‐derived factor 1 was higher in both HIF‐α‐expressing BMSCs, enhanced migration toward vascular endothelial growth factor was found only following overexpression of HIF‐2α, supported by a robust expression of its receptor, Flk‐1. HIF‐α expression was associated with upregulation of angiogenic proteins and improved tube formation. Cytokine arrays of endothelial cells stimulated by medium collected from HIF‐α‐expressing BMSCs revealed further angiogenic activation and improved adhesive capacity. Eventually, delivery of HIF‐2α‐transduced BMSCs induced a more robust angiogenic response, compared with sham‐transduced or HIF‐1α‐transduced BMSCs in the corneal micropocket angiogenesis model. Our results support the use of HIF‐α genes, particularly HIF‐2α, to augment the efficacy of future cell‐based therapy.

Retinal ganglion cell apoptotic pathway in glaucoma: Initiating and downstream mechanisms

Apoptosis of retinal ganglion cells (RGCs) in glaucoma causes progressive visual field loss, making it the primary cause of irreversible blindness worldwide. Elevated intraocular pressure and aging, the main risk factors for glaucoma, accelerate RGC apoptosis. Numerous pathways and mechanisms were found to be involved in RGC death in glaucoma. Neurotrophic factors deprivation is an early event. Oxidative stress, mitochondrial dysfunction, inflammation, glial cell dysfunction, and activation of apoptotic pathways and prosurvival pathways play a significant role in RGC death in glaucoma. The most important among the involved pathways are the MAP-kinase pathway, PI-3 kinase/Akt pathway, Bcl-2 family, caspase family, and IAP family.

Solar Exposure and Residential Geographic History in Relation to Exfoliation Syndrome in the United States and Israel

IMPORTANCE Residential (geographic) history and extent of solar exposure may be important risk factors for exfoliation syndrome (XFS) but, to our knowledge, detailed lifetime solar exposure has not been previously evaluated in XFS. OBJECTIVE To assess the relation between residential history, solar exposure, and XFS. DESIGN, SETTING, AND PARTICIPANTS This clinic-based case-control study was conducted in the United States and Israel. It involved XFS cases and control individuals (all ≥ 60-year-old white individuals) enrolled from 2010 to 2012 (United States: 118 cases and 106 control participants; Israel: 67 cases and 72 control participants). MAIN OUTCOMES AND MEASURES Weighted lifetime average latitude of residence and average number of hours per week spent outdoors as determined by validated questionnaires. RESULTS In multivariable analyses, each degree of weighted lifetime average residential latitude away from the equator was associated with 11% increased odds of XFS (pooled odds ratio [OR], 1.11; 95% CI, 1.05-1.17; P < .001). Furthermore, every hour per week spent outdoors during the summer, averaged over a lifetime, was associated with 4% increased odds of XFS (pooled OR, 1.04; 95% CI, 1.00-1.07; P = .03). For every 1% of average lifetime summer time between 10 am and 4 pm that sunglasses were worn, the odds of XFS decreased by 2% (OR, 0.98; 95% CI, 0.97-0.99; P < .001) in the United States but not in Israel (OR, 1.00; 95% CI, 0.99-1.01; P = .92; P for heterogeneity = .005). In the United States, after controlling for important environmental covariates, history of work over water or snow was associated with increased odds of XFS (OR, 3.86; 95% CI, 1.36-10.9); in Israel, there were too few people with such history for analysis. We did not identify an association between brimmed hat wear and XFS (P > .57). CONCLUSIONS AND RELEVANCE Lifetime outdoor activities may contribute to XFS. The association with work over snow or water and the lack of association with brimmed hat wear suggests that ocular exposure to light from reflective surfaces may be an important type of exposure in XFS etiology.

Drug Modification of Angiogenesis in a Rat Cornea Model

PURPOSE To evaluate the influence of some widely used antiglaucoma agents on angiogenesis in a novel rat cornea model. METHODS Angiogenesis was induced in 32 rats by slow-release polymer pellets containing basic fibroblast growth factor (bFGF) placed in a corneal micropocket. Angiogenesis was later measured and compared in groups of rats given one of four antiglaucoma drug therapies and one control group. The drugs were commercially available preparations of prostaglandins, beta-blockers, alpha-2 agonists, and carbonic anhydrase inhibitors given for 7 days in a manner similar to that used in humans. Growth was measured by calculating the maximum linear vessel growth divided by pellet-limbus distance. RESULTS Biomicroscopic observation disclosed that all tested animals showed an induction of neovascular reactions in their corneal stroma. The growth index results for the control, latanoprost, dorzolamide, brimonidine, and timolol malate groups were 1.65 +/- 0.16, 1.98 +/- 0.18, 1.85 +/- 0.19, 2.03 +/- 0.38, and 1.65 +/- 0.14, respectively, confirming the hypothesis that topically delivered antiglaucoma drugs modify the normal angiogenic response. Of them, the prostaglandins showed the most prominent angiogenic stimulatory effect (P = 0.03). CONCLUSIONS This modified micropocket assay of corneal angiogenesis in rats demonstrated the stimulatory effect of several widely used topically delivered antiglaucoma medications on the angiogenic process. The results indicate that the selection of drugs for treating different ophthalmic diseases should take into account their influence on angiogenic processes.

Epiretinal transplantation of human bone marrow mesenchymal stem cells rescues retinal and vision function in a rat model of retinal degeneration

Vision incapacitation and blindness associated with incurable retinal degeneration affect millions of people worldwide. In this study, 0.25×10(6) human bone marrow stem cells (hBM-MSCs) were transplanted epiretinally in the right eye of Royal College Surgeons (RCS) rats at the age of 28 days. Epiretinally transplanted cells were identified as a thin layer of cells along vitreous cavity, in close proximity to the retina or attached to the lens capsule, up to 6 weeks following transplantation. Epiretinal transplantation delayed photoreceptor degeneration and rescued retinal function up to 20 weeks following cell transplantation. Visual functions remained close to normal levels in epiretinal transplantation rats. No inflammation or any other adverse effects were observed in transplanted eyes. Our findings suggest that transplantation of hBM-MSCs as a thin epiretinal layer is effective for treatment of retinal degeneration in RCS rats, and that transplanting the cells in close proximity to the retina enhances hBM-MSC therapeutic effect compared with intravitreal injection.

Regulation of Cell Death and Survival Pathways in Secondary Degeneration of the Optic Nerve – A Long-Term Study

Purpose: To investigate cell death and survival pathways in secondary degeneration of the optic nerve (ON) and retina over a period of 6 months. Methods: A partial transection model of the ON that morphologically separates primary and secondary degeneration was applied unilaterally in 89 Wistar rat eyes. The upper third of the retinas were analyzed for primary degeneration, while the lower third of the retinas were analyzed for secondary degeneration. The involvement of members of the mitogen-activated protein (MAP) kinase pathway and the PI-3-kinase/Akt pathway were evaluated in primary and secondary degeneration in multiple time points over a period of 6 months using immunohistochemistry and western blotting. Results were compared to corresponding areas from control fellow eyes. Results: All investigated members of the MAP kinase pathway were significantly activated in primary degeneration, secondary degeneration or both. P-SAPK/JNK and P-ERK were activated in primary degeneration without a concomitant activation in secondary degeneration. The prosurvival protein p-Akt, a member of the PI-3-kinase survival pathway, was significantly activated in secondary but not in primary degeneration. P-c-jun and p-ATF were significantly activated in both primary and secondary degeneration. The time-dependent pattern of activation was different for each protein and in secondary degeneration the activation of these proteins was usually short termed. Conclusions: The significant involvement of the MAP kinase pathway and the PI-3-kinase survival pathway in secondary degeneration of the ON and retina is short termed despite continuous retinal ganglion cells (RGCs)apoptosis for at least 6 months.

Ranibizumab for persistent diabetic macular edema after bevacizumab treatment

Purpose To evaluate the efficacy of switching from bevacizumab to ranibizumab in patients with diabetic macular edema (DME). Methods This was a retrospective study of patients with DME initially treated with bevacizumab and switched to ranibizumab. Visual acuity (VA) and central retinal thickness (CRT) were retrieved at fixed timepoints prior to and after the switch. Results Forty eyes of 32 patients were included in the study. The difference in VA between any of these fixed timepoints was not statistically significant. A significant gain in VA was found in eyes that lost more than 0.1 logMAR during treatment with the last 3 bevacizumab injections. The mean CRT was significantly lower after the first 3 ranibizumab injections and at the final follow-up (p<0.001), a 67 ± 14 μm and 78 ± 18 μm reduction in thickness, respectively. Conclusions Switching to ranibizumab resulted in a significant decrease in the CRT of eyes with DME, and should be considered when there is a lack of response or deterioration while on bevacizumab injections. A significant gain in VA was observed in a subgroup of eyes that lost more than one line while receiving the last 3 bevacizumab injections prior to the switch.