Dr. Kaufman is a Professor in the UW Department of Ophthalmology and Visual Sciences at the University of Wisconsin School of Medicine and Public Health. His research centers on studies of the physiology, pharmacology, morphology, cell biology, genetic manipulation, neural control, biomechanics and aging of the aqueous humor formation and drainage and accommodative mechanisms in the non-human primate, seeking to understand the pathophysiology and develop new therapies for the human diseases of glaucoma and presbyopia.
Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms
Invest Ophthalmol Vis Sci. 2022 Feb 1;63(2):12. doi: 10.1167/iovs.63.2.12.
Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.
Report: The effects of topical pleurotus tuberregium (PT) aqueous extract on intraocular pressure in monkeys
PLoS One. 2021 Aug 24;16(8):e0256422. doi: 10.1371/journal.pone.0256422. eCollection 2021.
PURPOSE: In earlier experiments in Nigeria, aqueous extract of Pleurotus tuber-regium (PT) had been shown to lower intra ocular pressure (IOP) in a feline model. The aim of the current study was to determine whether PT had the same or a similar IOP-lowering effect in ocularly normal non-human primates.
METHODS: Four monkeys were treated twice daily for 4 days with 2 x 20 μl drops of 50 mg/ml PT (pH = 4.3). The monkeys were sedated with 5-10 mg/kg ketamine HCl IM. PT was administered to the right eye and BSS to the left eye. Baseline IOP was measured just prior to beginning treatment, and on day 5 before treatment and then hourly for 3 hours, beginning 1 hour after treatment. SLEs were performed at baseline and on day 5 pre- and 3 hours post-treatment.
RESULTS: There was no significant difference between IOP in treated vs control eyes in the protocol. There were no adverse effects or toxicity as seen by SLE.
CONCLUSIONS: The inability of the extract to lower IOP in monkeys, in contrast to ocular hypertensive cats in an earlier study, could be due to species differences or duration of treatment. Since no adverse effects were observed in the monkeys, further studies with varying durations and dosages are recommended.
Deconstructing aqueous humor outflow - The last 50 years
Exp Eye Res. 2020 Aug;197:108105. doi: 10.1016/j.exer.2020.108105. Epub 2020 Jun 23.
Herein partially summarizes one scientist-clinician's wanderings through the jungles of primate aqueous humor outflow over the past ~45 years. Totally removing the iris has no effect on outflow facility or its response to pilocarpine, whereas disinserting the ciliary muscle (CM) from the scleral spur/trabecular meshwork (TM) completely abolishes pilocarpine's effect. Epinephrine increases facility in CM disinserted eyes. Cytochalasins and latrunculins increase outflow facility, subthreshold doses of cytochalasins and epinephrine given together increase facility, and phalloidin, which has no effect on facility, partially blocks the effect of both cytochalasins and epinephrine. H-7, ML7, Y27632 and nitric oxide - donating compounds all increase facility, consistent with a mechanosensitive TM/SC. Adenosine A1 agonists increase and angiotensin II decrease facility. OCT and optical imaging techniques now permit visualization and digital recording of the distal outflow pathways in real time. Prostaglandin (PG) F2α analogues increase the synthesis and release of matrix metalloproteinases by the CM cells, causing remodeling and thinning of the interbundle extracellular matrix (ECM), thereby increasing uveoscleral outflow and reducing IOP. Combination molecules (one molecule, two or more effects) and fixed combination products (two molecules in one bottle) simplify drug regimens for patients. Gene and stem cell therapies to enhance aqueous outflow have been successful in laboratory models and may fill an unmet need in terms of patient compliance, taking the patient out of the delivery system. Functional transfer of genes inhibiting the rho cascade or decoupling actin from myosin increase facility, while genes preferentially expressed in the glaucomatous TM decrease facility. In live NHP, reporter genes are expressed for 2+ years in the TM after a single intracameral injection, with no adverse reaction. However, except for one recent report, injection of facility-effective genes in monkey organ cultured anterior segments (MOCAS) have no effect in live NHP. While intracameral injection of an FIV. BOVPGFS-myc.GFP PGF synthase vector construct reproducibly induces an ~2 mmHg reduction in IOP, the effect is much less than that of topical PGF2⍺ analogue eyedrops, and dissipates after 5 months. The turnoff mechanism has yet to be defeated, although proteasome inhibition enhances reporter gene expression in MOCAS. Intracanalicular injection might minimize off-target effects that activate turn-off mechanisms. An AD-P21 vector injected sub-tenon is effective in 'right-timing' wound healing after trabeculectomy in live laser-induced glaucomatous monkeys. In human (H)OCAS, depletion of TM cells by saponification eliminates the aqueous flow response to pressure elevation, which can be restored by either cultured TM cells or by IPSC-derived TM cells. There were many other steps along the way, but much was accomplished, biologically and therapeutically over the past half century of research and development focused on one very small but complex ocular apparatus. I am deeply grateful for this award, named for a giant in our field that none of us can live up to.
Early adoption of the fluocinolone acetonide (FAc) intravitreal implant in patients with persistent or recurrent diabetic macular edema (DME)
Int Med Case Rep J. 2019 Apr 11;12:93-102. doi: 10.2147/IMCRJ.S191171. eCollection 2019.
Objective: To assess long-term outcomes for effectiveness, safety, and treatment burden after injection of 0.2 µg/day fluocinolone acetonide [FAc] intravitreal implant (ILUVIEN®) in patients with persistent or recurrent diabetic macular edema (DME) and 6-18 months of follow-up. Methods: Retrospective case series in 18 eyes (13 patients) treated with the FAc implant. Prior to the implant, eyes were treated with an anti-VEGF therapy, dexamethasone implant, or focal or panretinal photocoagulation. Effectiveness outcomes included changes in visual acuity and macular edema. Safety outcomes included intraocular pressure (IOP) changes, IOP drugs, and IOP-related surgeries/interventions. Treatment burden was assessed by comparing the number of DME treatments before and after FAc implantation. Results: The FAc implant reduced macular volume in 16/18 (89%) eyes, with a statistically significant mean change of -1.33 mm3 (p=0.001). The average central retinal thickness reduction for all 18 eyes was statistically significant, decreasing from 444 µm at baseline to 359 µm after the FAc implant (p<0.001). In 90% of eyes, visual acuity was stable throughout the follow-up period, with increases or no worsening in Early Treatment Diabetic Retinopathy Study letter score. Although mean IOP was statistically higher after treatment, it was within the normal range at all timepoints, and most (83.3%) eyes remained in the IOP category 0-22 mmHg, and the number of IOP treatments required did not increase and no patients required IOP-lowering surgery. Treatment burden for DME was reduced after the implant was administered, with 56% of eyes not requiring any additional treatment. The average number of treatments was 1.3 in the 6 months after the FAc implant versus 4.6 in the 12 months preceding the implant. Conclusion: The FAc implant is an appropriate option to incorporate earlier in the DME treatment process, leading to positive long-term outcomes with an acceptable safety profile, and a reduced treatment burden for patients, and reduced clinical staff time.
SB772077B, A New Rho Kinase Inhibitor Enhances Aqueous Humour Outflow Facility in Human Eyes
Sci Rep. 2018 Oct 19;8(1):15472. doi: 10.1038/s41598-018-33932-8.
We investigated the effect of a new Rho kinase inhibitor, SB772077B (SB77) on aqueous outflow facility (OF) in human eyes using human organ-cultured anterior segment (HOCAS). IOP was monitored for 24 h post-treatment with either SB77 (0.1/10/50 µM) or vehicle after a stable baseline pressure. The hydrodynamic pattern of aqueous outflow was analysed by labelling outflow pathway with red fluorescent microspheres. The effect of SB77 on cell morphology, actin stress fibers, focal adhesions, ECM, status of RhoA activation and myosin light chain phosphorylation (p-MLC) were evaluated and compared with Y27632, by immunostaining using primary human trabecular meshwork (HTM) cells. Following 24 h treatment, SB77 increased OF by 16% at 0.1 µM (N = 6), 29% at 10 µM (N = 8; p = 0.018) and 39% at 50 µM (N = 8; p = 0.004) in human eyes. There was an overall increase in tracer quantity and in area along inner wall of Schlemm's canal. Treatment with SB77 showed no evidence of cytotoxicity and caused a significant reduction in the expression of fibrotic markers compared to Y27632. The present findings indicate that SB77 treatment was effective in enhancing OF and reducing fibrotic markers in an ex vivo model. Thus SB77 may be a potential clinical candidate for the management of glaucoma.
Effects of Lentivirus-Mediated C3 Expression on Trabecular Meshwork Cells and Intraocular Pressure
Invest Ophthalmol Vis Sci. 2018 Oct 1;59(12):4937-4944. doi: 10.1167/iovs.18-24978.
PURPOSE: We evaluated the effects of lentivirus-mediated exoenzyme C3 transferase (C3) expression on cultured primary human trabecular meshwork (HTM) cells in vitro, and on rat intraocular pressure (IOP).
METHODS: HTM cells were cultured and treated with lentivirus vectors expressing either green fluorescent protein (GFP) only (LV-GFP) or GFP and C3 together (LV-C3-GFP). Changes in cell morphology and actin stress fibers were assessed. The vectors were also injected into the anterior chamber of rats, and GFP expression was visualized by a Micron III Retinal Imaging Microscope in vivo and a fluorescence microscope ex vivo. Changes in rat IOP were monitored by using a rebound tonometer and the eyes were evaluated by slit lamp.
RESULTS: LV-mediated C3 expression induced morphologic changes in HTM cells. The cells became retracted and rounded. GFP expression in the anterior chamber angle of rats was observed in vivo from 8 days to 48 days after injection of LV-C3-GFP or LV-GFP. IOP was significantly decreased in the LV-C3-GFP group starting 3 days post injection, and lasting for at least 40 days, when compared to either the contralateral control eyes (the LV-GFP group) or the ipsilateral baseline before injection (P < 0.05). No obvious inflammatory signs were observed in either the LV-C3-GFP or LV-GFP groups.
CONCLUSIONS: LV-mediated C3 expression induced changes in morphology of cultured HTM cells. Intracameral injection of LV-C3-GFP lowered rat IOP for at least 40 days. No significant inflammatory reactions were observed in either the LV-C3-GFP or LV-GFP groups. This study supports the possible use of C3 gene therapy for the treatment of glaucoma.
Medical Management of Glaucoma in Exfoliation Syndrome
J Glaucoma. 2018 Jul;27 Suppl 1:S87-S90. doi: 10.1097/IJG.0000000000000920.
The treatment of glaucoma in exfoliation syndrome is similar to primary open-angle glaucoma. Frequently, exfoliation glaucoma (XFG) patients require early polytherapy with topical medications. Little emphasis has been placed on tailoring treatment specifically to XFG. New outflow enhancing agents with novel mechanisms of action, such as Rho Kinase inhibition, NO signaling (both recently FDA-approved drugs) and adenosine α1-receptor stimulation, act directly on the trabecular meshwork. These agents may prove to be effective in lowering intraocular pressure and perhaps altering the pathogenesis of XFG aid in the long-term management of this disease.
Proteasome Inhibition Increases the Efficiency of Lentiviral Vector-Mediated Transduction of Trabecular Meshwork
Invest Ophthalmol Vis Sci. 2018 Jan 1;59(1):298-310. doi: 10.1167/iovs.17-22074.
PURPOSE: To determine if proteasome inhibition using MG132 increased the efficiency of FIV vector-mediated transduction in human trabecular meshwork (TM)-1 cells and monkey organ-cultured anterior segments (MOCAS).
METHODS: TM-1 cells were pretreated for 1 hour with 0.5% dimethyl sulfoxide (DMSO; vehicle control) or 5 to 50 μM MG132 and transduced with FIV.GFP (green fluorescent protein)- or FIV.mCherry-expressing vector at a multiplicity of transduction (MOT) of 20. At 24 hours, cells were fixed and stained with antibodies for GFP, and positive cells were counted, manually or by fluorescence-activated cell sorting (FACS). Cells transduced with FIV.GFP particles alone were used as controls. The effect of 20 μM MG132 treatment on high- and low-dose (2 × 107 and 0.8 × 107 transducing units [TU], respectively) FIV.GFP transduction with or without MG132 was also evaluated in MOCAS using fluorescence microscopy. Vector genome equivalents in cells and tissues were quantified by quantitative (q)PCR on DNA.
RESULTS: In the MG132 treatment groups, there was a significant dose-dependent increase in the percentage of transduced cells at all concentrations tested. Vector genome equivalents were also increased in TM-1 cells treated with MG132. Increased FIV.GFP expression in the TM was also observed in MOCAS treated with 20 μM MG132 and the high dose of vector. Vector genome equivalents were also significantly increased in the MOCAS tissues. Increased transduction was not seen with the low dose of virus.
CONCLUSIONS: Proteasome inhibition increased the transduction efficiency of FIV particles in TM-1 cells and MOCAS and may be a useful adjunct for delivery of therapeutic genes to the TM by lentiviral vectors.
Latanoprostene Bunod 0.024% in Subjects With Open-angle Glaucoma or Ocular Hypertension: Pooled Phase 3 Study Findings
J Glaucoma. 2018 Jan;27(1):7-15. doi: 10.1097/IJG.0000000000000831.
PURPOSE: To compare the diurnal intraocular pressure (IOP)-lowering effect of latanoprostene bunod (LBN) 0.024% with timolol maleate 0.5% in subjects with open-angle glaucoma (OAG) or ocular hypertension (OHT).
PATIENTS AND METHODS: Pooled analysis of two phase 3, randomized, multicenter, double-masked, parallel-group, noninferiority trials (APOLLO and LUNAR), each with open-label safety extension phases. Adults with OAG or OHT were randomized 2:1 to double-masked treatment with LBN once daily (qd) or timolol twice daily (bid) for 3 months followed by open-label LBN treatment for 3 (LUNAR) or 9 (APOLLO) months. IOP was measured at 8 AM, 12 PM, and 4 PM at week 2, week 6, and months 3, 6, 9, and 12.
RESULTS: Of the 840 subjects randomized, 774 (LBN, n=523; timolol crossover to LBN, n=251) completed the efficacy phase, and 738 completed the safety extension phase. Mean IOP was significantly lower with LBN versus timolol at all 9 evaluation timepoints during the efficacy phase (P<0.001). A significantly greater proportion of LBN-treated subjects attained a mean IOP ≤18 mm Hg and IOP reduction ≥25% from baseline versus timolol-treated subjects (P<0.001). The IOP reduction with LBN was sustained through the safety phase; subjects crossed over from timolol to LBN experienced additional significant IOP lowering (P≤0.009). Both treatments were well tolerated, and there were no safety concerns with long-term LBN treatment.
CONCLUSIONS: In this pooled analysis of subjects with OAG and OHT, LBN 0.024% qd provided greater IOP-lowering compared with timolol 0.5% bid and maintained lowered IOP through 12 months. LBN demonstrated a safety profile comparable to that of prostaglandin analogs.
Change in Diabetic Retinopathy Through 2 Years: Secondary Analysis of a Randomized Clinical Trial Comparing Aflibercept, Bevacizumab, and Ranibizumab
JAMA Ophthalmol. 2017 Jun 1;135(6):558-568. doi: 10.1001/jamaophthalmol.2017.0821.
IMPORTANCE: Anti-vascular endothelial growth factor (anti-VEGF) therapy for diabetic macular edema (DME) favorably affects diabetic retinopathy (DR) improvement and worsening. It is unknown whether these effects differ across anti-VEGF agents.
OBJECTIVE: To compare changes in DR severity during aflibercept, bevacizumab, or ranibizumab treatment for DME.
DESIGN, SETTING, AND PARTICIPANTS: Preplanned secondary analysis of data from a comparative effectiveness trial for center-involved DME was conducted in 650 participants receiving aflibercept, bevacizumab, or ranibizumab. Retinopathy improvement and worsening were determined during 2 years of treatment. Participants were randomized in 2012 through 2013, and the trial concluded on September 23, 2015.
INTERVENTIONS: Random assignment to aflibercept, 2.0 mg; bevacizumab, 1.25 mg; ranibizumab, 0.3 mg, up to every 4 weeks through 2 years following a retreatment protocol.
MAIN OUTCOMES AND MEASURES: Percentages with retinopathy improvement at 1 and 2 years and cumulative probabilities for retinopathy worsening through 2-year without adjustment for multiple outcomes.
RESULTS: A total of 650 participants (495 [76.2%] nonproliferative DR [NPDR], 155 proliferative DR [PDR]) were analyzed; 302 (46.5%) were women and mean (SD) age was 61 (10) years; 425 (65.4%) were white. At 1 year, among 423 NPDR eyes, 44 of 141 (31.2%) treated with aflibercept, 29 of 131 (22.1%) with bevacizumab, and 57 of 151 (37.7%) with ranibizumab had improvement of DR severity (adjusted difference: 11.7%; 95% CI, 2.9% to 20.6%; P = .004 for aflibercept vs bevacizumab; 8.9%; 95% CI, 1.7% to 16.1%; P = .01 for ranibizumab vs bevacizumab; and 2.9%; 95% CI, -5.7% to 11.4%; P = .51 for aflibercept vs ranibizumab). At 2 years, 33 eyes (24.8%) in the aflibercept group, 25 eyes (22.1%) in the bevacizumab group, and 40 eyes (31.0%) in the ranibizumab group had DR improvement; no treatment group differences were identified. For 93 eyes with PDR at baseline, 1-year improvement rates were 75.9% for aflibercept, 31.4% for bevacizumab, and 55.2% for ranibizumab (adjusted difference: 50.4%; 95% CI, 26.8% to 74.0%; P < .001 for aflibercept vs bevacizumab; 20.4%; 95% CI, -3.1% to 44.0%; P = .09 for ranibizumab vs bevacizumab; and 30.0%; 95% CI, 4.4% to 55.6%; P = .02 for aflibercept vs ranibizumab). These rates and treatment group differences appeared to be maintained at 2 years. Despite the reduced numbers of injections in the second year, 66 (59.5%) of NPDR and 28 (70.0%) of PDR eyes that manifested improvement at 1 year maintained improvement at 2 years. Two-year cumulative rates for retinopathy worsening ranged from 7.1% to 10.2% and 17.2% to 26.4% among anti-VEGF groups for NPDR and PDR eyes, respectively. No statistically significant treatment differences were noted.
CONCLUSIONS AND RELEVANCE: At 1 and 2 years, eyes with NPDR receiving anti-VEGF treatment for DME may experience improvement in DR severity. Less improvement was demonstrated with bevacizumab at 1 year than with aflibercept or ranibizumab. Aflibercept was associated with more improvement at 1 and 2 years in the smaller subgroup of participants with PDR at baseline. All 3 anti-VEGF treatments were associated with low rates of DR worsening. These data provide additional outcomes that might be considered when choosing an anti-VEGF agent to treat DME.
Latanoprostene bunod ophthalmic solution 0.024% for IOP lowering in glaucoma and ocular hypertension
Expert Opin Pharmacother. 2017 Mar;18(4):433-444. doi: 10.1080/14656566.2017.1293654. Epub 2017 Feb 20.
Intraocular pressure (IOP)-lowering has been demonstrated to slow the progression or onset of visual field loss in open-angle glaucoma (OAG) or ocular hypertension (OHT). Pharmacological lowering of IOP is the most common initial intervention in patients with OAG or OHT, however, many patients will require more than one therapy to achieve target IOP. Latanoprostene bunod is a novel nitric oxide (NO)-donating prostaglandin F2α analog for the reduction of IOP. Areas covered: Current knowledge concerning the mechanism of action of latanoprostene bunod is presented. Additionally, clinical safety and efficacy data from published Phase 1 (KRONUS), Phase 2 (VOYAGER, CONSTELLATION) and Phase 3 (APOLLO, LUNAR, JUPITER) studies are reviewed. Expert opinion: Latanoprostene bunod is a dual mechanism, dual pathway molecule, consisting of latanoprost acid, which is known to enhance uveoscleral (unconventional) outflow by upregulating matrix metalloproteinase expression and remodeling of the ciliary muscle's extracellular matrix, linked to an NO-donating moiety, which enhances trabecular meshwork/Schlemm's canal (conventional) outflow by inducing cytoskeletal relaxation via the soluble guanylyl cyclase-cyclic guanosine monophosphate (sGC-cGMP) signaling pathway. Latanoprostene bunod 0.024% solution applied topically once daily appears more effective in reducing IOP in OHT and OAG subjects than either latanoprost or timolol, with a side effect profile similar to that of latanoprost.
Age-related posterior ciliary muscle restriction - A link between trabecular meshwork and optic nerve head pathophysiology
Exp Eye Res. 2017 May;158:187-189. doi: 10.1016/j.exer.2016.07.007. Epub 2016 Jul 22.
The ciliary muscle plays a major role in controlling both accommodation and outflow facility in primates. The ciliary muscle and the choroid functionally form an elastic network that extends from the trabecular meshwork all the way to the back of the eye and ultimately attaches to the elastic fiber ring that surrounds the optic nerve and to the lamina cribrosa through which the nerve passes. The ciliary muscle governs the accommodative movement of the elastic network. With age ciliary muscle mobility is restricted by progressively inelastic posterior attachments and the posterior restriction makes the contraction progressively isometric; placing increased tension on the optic nerve region. In addition, outflow facility also declines with age and limbal corneoscleral contour bows inward. Age-related loss in muscle movement and altered limbal corneoscleral contour could both compromise the basal function of the trabecular meshwork. Further, recent studies in non-human primates show that the central vitreous moves posteriorly all the way back to the optic nerve region, suggesting a fluid current and a pressure gradient toward the optic nerve. Thus, there may be pressure and tension spikes on the optic nerve region during accommodation and these pressure and tension spikes may increase with age. This constellation of events could be relevant to glaucomatous optic neuropathy. In summary, our hypothesis is that glaucoma and presbyopia may be literally linked to each other, via the choroid, and that damage to the optic nerve may be inflicted by accommodative intraocular pressure and choroidal tension "spikes", which may increase with age.
Effects of Latanoprost and Bimatoprost on the Expression of Molecules Relevant to Ocular Inflow and Outflow Pathways
PLoS One. 2016 Mar 24;11(3):e0151644. doi: 10.1371/journal.pone.0151644. eCollection 2016.
BACKGROUND AND PURPOSE: The intraocular pressure (IOP)-lowering and side effects in response to different prostaglandin F2α analogues can be variable, but, the underlying basis for this difference remains unknown. This study investigated the differential changes of cellular proteins relevant to IOP-lowering effects of latanoprost and bimatoprost.
METHODS: The human T lymphoblast (MOLT-3) cell line and immortalized human trabecular meshwork (iHTM) cells were studied by quantitative PCR and by immunofluorescence after treatment with either latanoprost or bimatoprost. New Zealand white rabbit eyes were treated topically with each agent and, following euthanasia, anterior segment tissues were studied with immunostaining.
RESULTS: In cultured MOLT-3 cells, mRNA expression of both c-fos and matrix metalloproteinase 9 increased significantly in response to each agent. In addition, there was little change in tissue inhibitor of metalloproteinase (TIMP)-3 mRNA, but a significant decrease in TIMP-4. Fibronectin mRNA in MOLT-3 cells was down-regulated with bimatoprost, but was up-regulated with latanoprost. Immunofluorescence analysis of iHTM cells showed that intracellular fibronectin was significantly decreased by bimatoprost, but was increased by latanoprost. Both latanoprost and bimatoprost increased mRNA expression of NF-кB p65 and decreased that of IкBα. Aquaporin-1 mRNA expression was significantly down-regulated by bimatoprost. Immunostaining also revealed a significant decrease of aquaporin-1 in the ciliary epithelium of New Zealand white rabbits after bimatoprost treatment.
CONCLUSIONS: Similarities in protein expression produced by latanoprost and bimatoprost in vitro may be relevant to the mechanism for their IOP-lowering effects in vivo. Differences in fibronectin expression and in aquaporin-1 expression in response to each agent may contribute to variability in the IOP-lowering efficacy in some studies.
Accommodative movements of the lens/capsule and the strand that extends between the posterior vitreous zonule insertion zone & the lens equator, in relation to the vitreous face and aging
Ophthalmic Physiol Opt. 2016 Jan;36(1):21-32. doi: 10.1111/opo.12256.
PURPOSE: To elucidate the dynamic accommodative movements of the lens capsule, posterior lens and the strand that attaches to the posterior vitreous zonule insertion zone and posterior lens equator (PVZ INS-LE), and their age-related changes.
METHODS: Twelve human subjects (ages 19-65 years) and 12 rhesus monkeys (ages 6-27 years) were studied. Accommodation was induced pharmacologically (humans) or by central electrical stimulation (monkeys). Ultrasound biomicroscopy was used to image intraocular structures in both species. Surgical procedures and contrast agents were utilized in the monkey eyes to elucidate function and allow visualization of the intraocular accommodative structures.
RESULTS: Human: The posterior pole of the lens moves posteriorly during accommodation in proportion to accommodative amplitude and ciliary muscle movement. Monkey: Similar accommodative movements of the posterior lens pole were seen in the monkey eyes. Following extracapsular lens extraction (ECLE), the central capsule bows backward during accommodation in proportion to accommodative amplitude and ciliary muscle movement, while the peripheral capsule moves forward. During accommodation the ciliary muscle moved forward by ~1.0 mm, pulling forward the vitreous zonule and the PVZ INS-LE structure. During the accommodative response the PVZ INS-LE structure moved forward when the lens was intact and when the lens substance and capsule were removed. In both the monkey and the human eyes these movements declined with age.
CONCLUSIONS: The accommodative shape change of the central capsule may be due to the elastic properties of the capsule itself. For these capsule/lens accommodative posterior movements to occur, the vitreous face must either allow for it or facilitate it. The PVZ INS-LE structure may act as a 'strut' to the posterior lens equator (pushing the lens equator forward) and thereby facilitate accommodative forward lens equator movement and lens thickening. The age-related posterior restriction of the ciliary muscle, vitreous zonule and the PVZ-INS LE structure dampens the accommodative lens shape change. Future descriptions of the accommodative mechanism, and approaches to presbyopia therapy, may need to incorporate these findings.
Anteriorly located zonular fibres as a tool for fine regulation in accommodation
Ophthalmic Physiol Opt. 2016 Jan;36(1):13-20. doi: 10.1111/opo.12257. Epub 2015 Oct 21.
PURPOSE: To describe an anteriorly located system of zonular fibres that could be involved in fine-tuning of accommodation.
METHODS: Forty-six human and 28 rhesus monkey eyes were dissected and special preparations were processed for scanning electron microscopy and reflected-light microscopy. Additional series of frontal and sagittal histological and ultrathin sections were analysed in respect to the origin and insertion of anteriorly located zonules. The presence of sensory terminals at the site of the originating zonules within the connective tissue of the ciliary body was studied by immunohistochemistry. For in-vivo visualization ultrasound biomicroscopy (UBM) was performed on 12 human subjects.
RESULTS: Fine zonular fibres originated from the valleys and lateral walls of the most anterior pars plicata that covers the anterior and inner circular ciliary muscle portion. These most anterior zonules (MAZ) showed attachments either to the anterior or posterior tines or they inserted directly onto the surface of the lens. At the site of origin, the course of the MAZ merged into the connective tissue fibres connecting the adjacent pigmented epithelium to the ciliary muscle. Numerous afferent terminals directly at the site of this MAZ-origin were connected to the intrinsic nervous network of the ciliary muscle.
CONCLUSIONS: A newly described set of zonular fibres features the capabilities to register the tensions of the zonular fork and lens capsule. The close location and neural connection towards the circular ciliary muscle portion could provide the basis for stabilization and readjustment of focusing that serves fast and fine-tuned accommodation and disaccommodation.
Effect of topical latanoprost 0.005% on intraocular pressure and pupil diameter in normal and glaucomatous cats
Vet Ophthalmol. 2016 Jul;19 Suppl 1(Suppl 1):13-23. doi: 10.1111/vop.12292. Epub 2015 Jul 16.
OBJECTIVE: To determine the effects of latanoprost on intraocular pressure (IOP) and pupil diameter (PD) in cats with inherited primary congenital glaucoma (PCG) and normal cats.
ANIMALS STUDIED AND PROCEDURES: IOP and PD were measured in both eyes (OU) of 12 adult cats (six normal, six PCG), three times per week for 3 weeks prior to, for 3 weeks during, and for 2 weeks following twice-daily treatment with 0.005% latanoprost to the right eye (OD) and vehicle to the left (control) eye (OS). IOP and PD were measured hourly, for 8 h, 1 day prior to, and on the first and last days of treatment. Aqueous humor flow rate (AHF) was determined at baseline and at the end of the treatment phase in six normal cats.
RESULTS: Mean IOP was significantly lower in treated vs. control eyes of PCG cats, for up to 8 h following a single latanoprost treatment, and a maximal IOP reduction of 63% occurred in treated eyes at 3 h. Latanoprost acutely lowered IOP in cats with PCG, but this effect appeared to diminish over 3 weeks of treatment. AHF was modestly increased in the treated eyes of normal cats after 3 weeks of latanoprost treatment, although IOP was not significantly affected. Latanoprost caused miosis, with rebound mydriasis at 24 h posttreatment, in the treated eyes of all cats.
CONCLUSIONS: Further research is needed to determine the suitability and efficacy of latanoprost treatment for long-term IOP-lowering in cats with PCG or other forms of glaucoma.
Rat, mouse, and primate models of chronic glaucoma show sustained elevation of extracellular ATP and altered purinergic signaling in the posterior eye
Invest Ophthalmol Vis Sci. 2015 May;56(5):3075-83. doi: 10.1167/iovs.14-15891.
PURPOSE: The cellular mechanisms linking elevated IOP with glaucomatous damage remain unresolved. Mechanical strains and short-term increases in IOP can trigger ATP release from retinal neurons and astrocytes, but the response to chronic IOP elevation is unknown. As excess extracellular ATP can increase inflammation and damage neurons, we asked if sustained IOP elevation was associated with a sustained increase in extracellular ATP in the posterior eye.
METHODS: No ideal animal model of chronic glaucoma exists, so three different models were used. Tg-Myoc(Y437H) mice were examined at 40 weeks, while IOP was elevated in rats following injection of hypertonic saline into episcleral veins and in cynomolgus monkeys by laser photocoagulation of the trabecular meshwork. The ATP levels were measured using the luciferin-luciferase assay while levels of NTPDase1 were assessed using qPCR, immunoblots, and immunohistochemistry.
RESULTS: The ATP levels were elevated in the vitreal humor of rats, mice, and primates after a sustained period of IOP elevation. The ecto-ATPase NTPDase1 was elevated in optic nerve head astrocytes exposed to extracellular ATP for an extended period. NTPDase1 was also elevated in the retinal tissue of rats, mice, and primates, and in the optic nerve of rats, with chronic elevation in IOP.
CONCLUSIONS: A sustained elevation in extracellular ATP, and upregulation of NTPDase1, occurs in the posterior eye of rat, mouse, and primate models of chronic glaucoma. This suggests the elevation in extracellular ATP may be sustained in chronic glaucoma, and implies a role for altered purinergic signaling in the disease.
Viral Vector Effects on Exoenzyme C3 Transferase-Mediated Actin Disruption and on Outflow Facility
Invest Ophthalmol Vis Sci. 2015 Apr;56(4):2431-8. doi: 10.1167/iovs.14-15909.
PURPOSE: Purified Clostridium botulinum exoenzyme C3 transferase (C3) effects on the actin cytoskeleton in human trabecular meshwork cells (HTM) and on the outflow facility response in monkey organ-cultured anterior segments (MOCAS) were determined in the presence or absence of viral vectors.
METHODS: Human adenovirus type 5 (AdV) and feline immunodeficiency virus (FIV) vectors were produced using kits. Cell soluble purified C3 (C3cs) was purchased commercially. Recombinant C3 (C3rec) cDNA was overexpressed in Escherichia coli and purified. The HTM cells were incubated with up to 10 μg/mL C3cs or with 5 μg of C3rec and/or viral vector (multiplicity of infection [MOI] = 25). Cells then were fixed and stained for actin. Outflow facility in MOCAS was measured at baseline, 4 hours, 24 hours, and 3 to 4 days following bolus injection of AdV (1.6 × 107 transducing units) and/or 2.5 μg C3rec.
RESULTS: The HTM cells treated for 4 hours with C3cs (all doses) or for 24 hours with C3rec developed a rounded morphology and lost stress fibers. Cells transduced with vectors alone showed no changes at any time point. Cells exposed to C3rec and cotransduced with either viral vector showed significant disruption of the actin cytoskeleton within 4 hours after exposure, which persisted at 24 hours. In MOCAS, the AdV vector alone had no effect on outflow facility, but enhanced the response to C3rec at 4 hours.
CONCLUSIONS: Coadministration of viral vectors enhances the ability of C3 transferase to disrupt actin stress fiber formation in HTM cells and increase outflow facility in MOCAS. Viral vectors potentially could be used to increase the bioavailability of proteins for cells that are difficult to transfect.
A randomised, controlled comparison of latanoprostene bunod and latanoprost 0.005% in the treatment of ocular hypertension and open angle glaucoma: the VOYAGER study
Br J Ophthalmol. 2015 Jun;99(6):738-45. doi: 10.1136/bjophthalmol-2014-305908. Epub 2014 Dec 8.
AIM: To assess the efficacy and safety of latanoprostene bunod (LBN) compared with latanoprost 0.005%, and to determine the optimum drug concentration(s) of LBN in reducing intraocular pressure (IOP) in subjects with open angle glaucoma or ocular hypertension.
METHODS: Randomised, investigator-masked, parallel-group, dose-ranging study. Subjects instilled one drop of study medication in the study eye once daily each evening for 28 days and completed five study visits. The primary efficacy endpoint was the reduction in mean diurnal IOP at Day 28.
RESULTS: Of the 413 subjects randomised (LBN 0.006%, n=82; LBN 0.012%, n=85; LBN 0.024%, n=83; LBN 0.040%, n=81; latanoprost, n=82), 396 subjects completed the study. Efficacy for LBN was dose-dependent reaching a plateau at 0.024%-0.040%. LBN 0.024% led to significantly greater reductions in diurnal IOP compared with latanoprost at the primary endpoint, Day 28 (p=0.005), as well as Days 7 (p=0.033) and 14 (p=0.015). The incidence of adverse events, mostly mild and transient, was numerically higher in the LBN treatment groups compared with the latanoprost group. Hyperaemia was similar across treatments.
CONCLUSIONS: LBN 0.024% dosed once daily was the lower of the two most effective concentrations evaluated, with significantly greater IOP lowering and comparable side effects relative to latanoprost 0.005%. LBN dosed once daily for 28 days was well tolerated.
CLINICAL TRIAL NUMBER: NCT01223378.
Morphological alterations within the peripheral fixation of the iris dilator muscle in eyes with pigmentary glaucoma
Invest Ophthalmol Vis Sci. 2014 Jun 17;55(7):4541-51. doi: 10.1167/iovs.13-13765.
PURPOSE: To analyze the peripheral fixation of the iris dilator muscle in normal eyes and in eyes with pigmentary glaucoma (PG).
METHODS: Using 63 control eyes (age 18 months-99 years), the peripheral iris dilator was investigated by light microscopy, immunohistochemistry, and electron microscopy. Development was studied using 18 differently aged fetal eyes stained immunohistochemically against α-smooth muscle (SM) actin. The peripheral iris dilator muscle in PG was analyzed using semithin and ultrathin sections of six glutaraldehyde-fixed eyes from three donors aged 38, 62, and 74 years.
RESULTS: In normal eyes, the peripheral end of the iris dilator muscle is arranged in a sphincter-like manner. Arcade-shaped tendinous connections associated with myofibroblasts (iridial strands) anchor the iris dilator within the elastic-fibromuscular ciliary meshwork that also serves as fixation area for the elastic tendons of the inner ciliary muscle portions. The iridial strands are innervated and can adapt their length during accommodation. The PG eyes show incomplete circular bundles and iridial strands that are mainly anchored to the iris stroma and the flexible uveal parts of the trabecular meshwork.
CONCLUSIONS: The normal anchorage of the peripheral iris dilator and its presumably neuronally regulated length adaptation stabilize the peripheral iris during accommodation. Insufficient fixation in PG could promote posterior bowing of the iris with rubbing against the zonular fibers and pigment liberation from the iris pigmented epithelium.
Neuroprotective effects of C3 exoenzyme in excitotoxic retinopathy
Exp Eye Res. 2014 Aug;125:128-34. doi: 10.1016/j.exer.2014.05.018. Epub 2014 Jun 11.
The purpose of this study is to evaluate the neuroprotective effects of C3 exoenzyme (C3) on N-methyl-d-aspartate (NMDA)-induced retinopathy in rats. C3 was expressed in Escherichia. coli and purified by affinity chromatography. Immunofluorescence was performed in NIH 3T3 cells treated with C3 to verify the cellular uptake of the protein. NMDA was injected intravitreally into rat eyes with or without C3. At various time points after injection, eyes were enucleated. Hematoxylin/eosin staining was performed on retina cross-sections for morphological analysis. Survival and apoptosis of cells in the ganglion cell layer (GCL) were assessed by cresyl violet staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) on retina flat-mounts. RhoA levels in retina cells were evaluated by Western blot to detect C3 uptake in vivo. The cellular uptake of C3 was verified by immunofluorescence. Damage including a decrease in inner plexiform layer (IPL) thickness and reduction of cell density in the GCL, corresponding to apoptosis of neurons, was induced by intravitreal injection of NMDA. Protection against this damage was observed following co-injection of C3 and NMDA. RhoA ADP-ribosylation induced by C3 was confirmed by Western blot. Our results suggest that C3 exerts neuroprotective effects against excitotoxic damage induced by NMDA.
A Potential Application of Canaloplasty in Glaucoma Gene Therapy
Transl Vis Sci Technol. 2013 Jan 31;2(1):2. doi: 10.1167/tvst.2.1.2.
Canaloplasty, a recently developed non-penetrating glaucoma surgical approach, may restore physiological outflow routes in primary open-angle glaucoma with less risk of severe postoperative complications than trabeculectomy. Since the inner wall of Schlemm's canal (SC) is directly in contact with the trabecular meshwork (TM) for 360 degrees and the catheter device used in canaloplasty allows viscoelastic to be injected into the entire length of SC, canaloplasty might also be used to perform SC/TM-targeted delivery of transgene vectors for glaucoma gene therapy. This hypothesized new method for transgene delivery may give the transgene access to the entire inner wall of SC and the whole juxtacanalicular region of the TM and allow the transgene to be expressed in both the TM and SC without affecting the cornea, iris and ciliary body. Further, this strategy might have a greater trabecular outflow resistance-decreasing effect than either the genetic or surgical approach alone.
Effect of nitric oxide on anterior segment physiology in monkeys
Invest Ophthalmol Vis Sci. 2013 Jul 30;54(7):5103-10. doi: 10.1167/iovs.12-11491.
PURPOSE: To determine the effect of the nitric oxide donor, sodium nitroprusside (SNP), and the nitric oxide synthase (NOS) inhibitor, L-nitro-arginine-methylester (L-NAME), on IOP, mean arterial pressure (MAP), pupil diameter (PD), refraction (Rfx), aqueous humor formation (AHF), and outflow facility (OF) in monkeys.
METHODS: Monkeys were treated with single or multiple topical treatments of 500 μg SNP or L-NAME to one eye. IOP was determined by Goldmann applanation tonometry, PD with vernier calipers in room light, Rfx by Hartinger coincidence refractometry, AHF by fluorophotometry, and MAP with a blood pressure monitor. OF was determined by two-level constant pressure perfusion following anterior chamber exchange.
RESULTS: Following four topical treatments with 500 μg SNP, 30 minutes apart, IOP was significantly decreased from 2 to 6 hours compared with the contralateral control with the maximum IOP reduction of 20% at 3 hours (P < 0.001). PD, Rfx, and AHF were unchanged. Effects on MAP were variable. OF after SNP exchange was significantly increased by 77% (P < 0.05) at 10(-3) M. Topical L-NAME had no effect on IOP, PD, Rfx, or MAP.
CONCLUSIONS: Enhancement of nitric oxide concentration at targeted tissues in the anterior segment may be a useful approach for IOP reduction for glaucoma therapy. Additional studies are warranted before conclusions can be made regarding the effect of NOS inhibition on ocular physiology in nonhuman primates.
Extralenticular and lenticular aspects of accommodation and presbyopia in human versus monkey eyes
Invest Ophthalmol Vis Sci. 2013 Jul 26;54(7):5035-48. doi: 10.1167/iovs.12-10846.
PURPOSE: To determine if the accommodative forward movements of the vitreous zonule and lens equator occur in the human eye, as they do in the rhesus monkey eye; to investigate the connection between the vitreous zonule posterior insertion zone and the posterior lens equator; and to determine which components-muscle apex width, lens thickness, lens equator position, vitreous zonule, circumlental space, and/or other intraocular dimensions, including those stated in the objectives above-are most important in predicting accommodative amplitude and presbyopia.
METHODS: Accommodation was induced pharmacologically in 12 visually normal human subjects (ages 19-65 years) and by midbrain electrical stimulation in 11 rhesus monkeys (ages 6-27 years). Ultrasound biomicroscopy imaged the entire ciliary body, anterior and posterior lens surfaces, and the zonule. Relevant distances were measured in the resting and accommodated eyes. Stepwise regression analysis determined which variables were the most important predictors.
RESULTS: The human vitreous zonule and lens equator move forward (anteriorly) during accommodation, and their movements decline with age, as in the monkey. Over all ages studied, age could explain accommodative amplitude, but not as well as accommodative lens thickening and resting muscle apex thickness did together. Accommodative change in distances between the vitreous zonule insertion zone and the posterior lens equator or muscle apex were important for predicting accommodative lens thickening.
CONCLUSIONS: Our findings quantify the movements of the zonule and ciliary muscle during accommodation, and identify their age-related changes that could impact the optical change that occurs during accommodation and IOL function.
Comparisons of actin filament disruptors and Rho kinase inhibitors as potential antiglaucoma medications
Expert Rev Ophthalmol. 2012 Apr;7(2):177-187. doi: 10.1586/eop.12.12.
Dynamics of the actin cytoskeleton in the trabecular meshwork play a crucial role in the regulation of trabecular outflow resistance. The actin filament disruptors and Rho kinase inhibitors affect the dynamics of the actomyosin system by either disrupting the actin filaments or inhibiting the Rho kinase-activated cellular contractility. Both approaches induce similar morphological changes and resistance decreases in the trabecular outflow pathway, and thus both have potential as antiglaucoma medications. Although the drugs might induce detrimental changes in the cornea following topical administration, lower drug concentrations in larger volumes as used clinically, but not higher drug concentrations in smaller volumes as used experimentally, could minimize corneal toxicity. Additionally, developments of trabecular meshwork-specific actin filament disruptors or Rho kinase inhibitors, prodrugs and new drug-delivery methods might avoid the drugs' toxicity to the cornea. Gene therapies with cytoskeleton-modulating proteins may mimic the effects of the cytoskeleton-modulating agents and have the potential to permanently decrease trabecular outflow resistance.
Cytoskeletal drugs prevent posterior capsular opacification in human lens capsule in vitro
Graefes Arch Clin Exp Ophthalmol. 2012 Apr;250(4):507-14. doi: 10.1007/s00417-011-1869-4. Epub 2011 Dec 3.
BACKGROUND: To determine whether the cytoskeletal drugs H-7 and Latrunculin B (LAT-B) inhibit posterior capsular opacification (PCO) in the cultured human lens capsular bag.
METHODS: Following extracapsular cataract (lens) extraction in human donor eyes, the capsular bag was prepared and cultured by standard techniques. Forty-eight capsular bags were studied, of which 13 were treated with H-7 (50, 100 or 300 μM), 12 with 1% BSS (vehicle of H-7), 11 with LAT-B (2, 5 or 10 μM), and 12 with 0.25% DMSO (vehicle of LAT-B). Forty out of the 48 capsular bags were from paired eyes of 20 donors, with one bag being treated with H-7/LAT-B and the other with BSS/DMSO for each pair, including 20 for the H-7-BSS protocol and 20 for the LAT-B-DMSO protocol. The medium with the cytoskeletal drug/vehicle was replaced every 3-4 days for 4 weeks. PCO was assessed daily using inverted phase-contrast microscopy, and scored on a 4-point scale.
RESULTS: In all cultures with BSS or DMSO, residual lens epithelial cells (LECs) on the anterior capsule migrated to and proliferated on the posterior capsule by 3-7 days, and apparent LEC growth on the posterior capsule with severe capsular wrinkling (PCO Grade 3) was seen by 2-3 weeks. When treated continuously with H-7 or LAT-B, the migration and proliferation of LECs and the capsular wrinkling were inhibited in a dose-dependent manner, with the inhibition being complete (PCO Grade 0) in the 300 μM H-7 (n = 8, p < 0.001) or 10 μM LAT-B culture (n = 3, p = 0.002).
CONCLUSION: H-7 and LAT-B dose-dependently inhibited PCO formation in the cultured human lens capsular bags, suggesting that cytoskeletal drugs might prevent PCO formation after surgery in the human eye.
Spectral domain OCT segmentation accuracy in monkeys
Investigative Ophthalmology & Visual Science, 51(13), pp.4401-4401
Interspecies and gender differences in multifocal electroretinograms of cynomolgus and rhesus macaques
Documenta ophthalmologica, 109, pp.73-86.