Lesya M. Shuba

Laminar Displacement and Prelaminar Tissue Thickness Change after Glaucoma Surgery Imaged with Optical Coherence Tomography

PURPOSE To study changes in lamina cribrosa position and prelaminar tissue thickness (PTT) after surgical IOP reduction in glaucoma patients. METHODS Twenty-two patients (mean age, 71.4 years) were imaged with spectral domain optical coherence tomography (SD-OCT; 24 radial B-scans centered on the optic nerve head [ONH]) before trabeculectomy or tube shunt implantation. Follow up images were acquired 1 week, 1 month, 3 months, and 6 months postsurgery. Bruchs membrane opening (BMO), the internal limiting membrane (ILM) and the anterior laminar surface (ALS) were segmented in each radial scan with custom software. Surfaces were fitted to the ILM and ALS with the extracted three-dimesional coordinates. PTT was the distance between the ILM and ALS, perpendicular to a BMO reference plane. Serial postsurgical laminar displacement (LD), relative to the BMO reference plane, and changes in PTT were measured. Positive values indicated anterior LD. RESULTS Mean (SD) presurgery IOP was 18.1 (6.5) mm Hg, and reduced by 4.7 (5.5), 2.4 (7.7), 7.0 (6.2), and 6.8 (7.5) mm Hg at 1 week, 1 month, 3 months, and 6 months postsurgery, respectively. At the four postsurgery time points, there was significant anterior LD (1.8 [9.5], -1.1 [8.9], 8.8 [20.2], and 17.9 [25.8] μm) and PTT increase (1.7 [13.3], 2.4 [11.9], 17.4 [13.7], and 13.9 [18.6] μm). LD was greater in ONHs with larger BMO area (P = 0.01) and deeper ALS (P = 0.04); however, PTT was not associated with any of the tested independent variables. CONCLUSIONS Both anterior LD and thickening of prelaminar tissue occur after surgical IOP reduction in patients with glaucoma.

Kinetic evidence distinguishing volume-sensitive chloride current from other types in guinea-pig ventricular myocytes.

1. Kinase‐mediated chloride currents (ICl) in guinea‐pig ventricular myocytes were activated by application of phorbol ester or forskolin, and compared with currents induced by hyposmotic swelling. Swelling‐activated current was identified as ICl from changes in reversal potential, outward rectification and conductance when the Cl‐gradient was modified. 2. Kinase‐stimulated currents were relatively time and voltage independent, whereas hyposmotic swelling‐stimulated (hyposmotic‐stimulated) currents inactivated during 100 ms pulses to positive potentials. Forskolin stimulated time‐independent ICl in myocytes with current unresponsive to hyposmotic superfusion, and superimposed a similar pedestal on time‐dependent ICl in swollen myocytes. 3. Less negative holding potentials depressed hyposmotic‐stimulated ICl tested at +80 mV; inhibition was half‐maximal at ‐25 mV. Pulses from ‐80 to +80 mV inactivated up to 75% of ICl along a multi‐exponential time course; repolarization elicited inwardly developing tail currents whose time courses suggest complex gating. 4. Hyperpolarizations, after strongly‐inactivating depolarizations, triggered reactivating tail currents whose amplitude and configuration were dependent on voltage and Cl‐gradients; tails were large and inwardly developing at potentials negative to the calculated Cl‐equilibrium potential (ECl), small and outwardly developing at potentials positive to ECl, and time independent near ECl. 5. These results suggest that the volume‐sensitive Cl‐ channels investigated here are distinct from other Cl‐ channels in guinea‐pig ventricular myocytes. However, their voltage‐dependent properties strongly resemble those of volume‐sensitive Cl‐ channels in certain epithelial cells.

Activation of cardiac chloride conductance by the tyrosine kinase inhibitor, genistein.

1 Genistein (GST), an inhibitor of protein tyrosine kinase (PTK), Na3VO4 (VO4), an inhibitor of phosphotyrosine phosphatase (PTPase), and forskolin (FSK), an activator of the cyclic AMP‐dependent, cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel, were applied to guinea‐pig ventricular myocytes to probe for a possible role of tyrosine phosphorylation in the regulation of cardiac Cl− channels. 2 Myocytes in the standard whole‐cell configuration were pulsed to various potentials and Cl− current (IC1) measured as the difference from control background current. GST (1–500 μm) activated a current that had similar biophysical properties (time‐ and voltage‐independent; Cl−‐dependent reversal potential and outward rectification) as IC1 activated by 5 μm FSK. The EC50 for activation of Cl− conductance (gC1) by GST was approximately 100 μm, and gC1 activated by GST (500 μm) was as large as gC1 activated by maximally‐effective FSK (5 μm). Daidzein, a GST analogue with little effect on PTK, was at least one order less effective than GST. 3 GST responses were rapidly and reversibly inhibited by 0.1‐1 mM VO4 treatments that had little effect on FSK‐activated IC1. 4 Niflumic acid (100–200 μm) reversibly depressed GST (100 μm)‐activated gC1 by 55%. 5 GST (50 μm) strongly incremented current in myocytes with cyclic AMP‐dependent CFTR IC1 already activated by maximally‐effective FSK 5 μm. 6 Based on these results, and on evidence of a synergistic interaction between GST and FSK, we conclude that inhibition of tyrosine phosphorylation by GST causes an activation of cardiac CFTR that is not mediated by an elevation of cyclic AMP.

Diurnal fluctuation and concordance of intraocular pressure in glaucoma suspects and normal tension glaucoma patients.

Purpose The study objective was to determine the concordance of intraocular pressure (IOP) in glaucoma suspects (GS) and normal tension glaucoma (NTG) patients. Methods This was a retrospective review of diurnal curves of untreated GS and NTG patients. No subject had IOP greater than 21 mm Hg. We defined GS patients as having suspicious optic nerves with normal visual fields, and NTG patients as having glaucomatous optic nerves with associated visual field loss. Goldmann applanation tonometry was performed at 10:00, 13:00, 16:00, 19:00, 22:00, and 07:00. Linear association of OD and OS IOP was estimated using Pearson correlation coefficient (r). The diurnal period was divided into 7 time intervals of 3, 6, 9, 12, 15, 18, and 21 hours, and the absolute difference in change in IOP between fellow eyes and probability that it was within 3 mm Hg were calculated. Results The study included 68 GS and 95 NTG subjects. The diurnal curves of the OD and OS showed a parallel course in both groups. The average correlations (r) of OD and OS IOP over the 6 time points were 0.78 and 0.81 for GS and NTG, respectively. The mean absolute difference in IOP change between OD and OS over the 6 time intervals ranged between 1.4 and 1.9 mm Hg for GS, and 1.3 and 1.5 mm Hg for NTG subjects. The probability that this difference was within 3 mm Hg ranged between 87% and 94% for GS, and 86% and 93% for NTG subjects. Conclusions The diurnal variation in IOP between the 2 eyes in GS and NTG is largely concordant in approximately 90% of the time.

Inhibition of the rapid component of the delayed-rectifier K+ current by therapeutic concentrations of the antispasmodic agent terodiline

1 Prolongation of the QT interval and malignant ventricular arrhythmia have been observed in patients administered terodiline for urinary incontinence. Since this adverse reaction might be caused by inhibition of delayed‐rectifier K+ current (IK), we investigated whether clinically relevant (10 μm) concentrations of the drug modify IK in guinea‐pig ventricular myocytes. 2 Myocytes superfused with normal Tyrodes solution were pulsed from −40 mV to more positive test potentials (V) for 0.2–1 s to elicit tail IK on repolarization and measure tail IK‐V relationships. IKr was distinguished from IKs by its sensitivity to the selective blocker E4031. 3 Inhibition of IKr by 5 μm E4031 was completely occluded by pretreatment with 3 μm terodiline. In addition, action potential lengthening by E4031 in guinea‐pig papillary muscles (29±3%) was abolished (3±2%) (P < 0.001) by terodiline pretreatment. 4 Inhibition of IKr by terodiline appeared to be voltage‐independent, and the parameters of the Hill equation describing the inhibition were IC50=0.7 μm and nH=1.6. High concentrations of the drug also affect IKs; in experiments with K+‐free Tyrodes, 10 μm terodiline inhibited tail IKs by 27±3% (n = 5) (P < 0.001). 5 These data suggest that QT lengthening at therapeutic concentrations of the drug (≈amp;1.5 μm) is primarily due to inhibition of IKr. Inhibition of other K+ currents such as IKs is likely to be important at higher concentrations.

Importance of Normal Aging in Estimating the Rate of Glaucomatous Neuroretinal Rim and Retinal Nerve Fiber Layer Loss

PURPOSE To describe longitudinal rates of change of neuroretinal parameters in patients with glaucoma and healthy controls, and to evaluate the influence of covariates. DESIGN Prospective longitudinal study. PARTICIPANTS Treated patients with glaucoma (n = 192) and healthy controls (n = 37). METHODS Global disc margin-based neuroretinal rim area (DMRA) was measured with confocal scanning laser tomography, while Bruchs membrane opening-minimum rim width (BMO-MRW), BMO area (BMOA), and peripapillary retinal nerve fiber layer thickness (RNFLT) were measured with optical coherence tomography at 6-month intervals. Individual rates of change were estimated with ordinary least-squares regression, and linear mixed effects modeling was used to estimate the average rate of change and differences between the groups, and to evaluate the effects of baseline measurement and baseline age on rates of change. MAIN OUTCOME MEASURES Rates of change for each parameter. RESULTS Subjects were followed for a median (range) of 4 (2-6) years. The proportion of controls who had significant reduction of neuroretinal parameters was 35% for BMO-MRW, 31% for RNFLT, and 11% for DMRA. The corresponding figures for patients with glaucoma were not statistically different (42%, P = 0.45; 31%, P = 0.99; 14%, P = 0.99, respectively). Controls had a significant reduction of BMO-MRW (mean: -1.92 μm/year, P < 0.01) and RNFLT (mean: -0.44 μm/year, P = 0.01), but not DMRA (mean: -0.22×10(-2) mm(2)/year, P = 0.41). After adjusting for covariates, patients with glaucoma had faster, but not statistically different, rates of deterioration compared with controls, by -1.26 μm/year (P = 0.07) for BMO-MRW, -0.40 μm/year (P = 0.11) for RNFLT, and -0.38×10(-2) mm(2)/year (P = 0.23) for DMRA. Baseline BMO-MRW and RNFLT significantly influenced the respective rates of change, with higher baseline values relating to faster reductions. Older age at baseline was associated with a slower reduction in rates of BMO-MRW. Reductions in intraocular pressure were related to increases in BMO-MRW and DMRA. There was a tendency for BMOA to decrease over time (-0.38×10(-2) mm(2)/year; P = 0.04). CONCLUSIONS Age-related loss of neuroretinal parameters may explain a large proportion of the deterioration observed in treated patients with glaucoma and should be carefully considered in estimating rates of change.

Correlation of capsular pseudoexfoliation material and iridocorneal angle pigment with the severity of pseudoexfoliation glaucoma.

PurposeTo evaluate the correlation between the amount of pseudoexfoliation (PXF) material on the anterior lens capsule, pigment in the iridocorneal angle, presenting intraocular pressure (IOP) and severity of glaucoma in patients with PXF glaucoma/syndrome. Patients and MethodsAnterior lens capsule PXF material and iridocorneal pigment of 98 untreated patients with PXF syndrome/glaucoma were graded from photographs and correlated with untreated IOP and indices of glaucoma severity (cup to disc ratio, and visual field mean deviation, and pattern standard deviation). ResultsThere was a positive statistically significant correlation between the iridocorneal angle pigmentation and IOP (P=0.047, R2=0.2), but not the indices of glaucoma severity (P>0.13). There was no significant correlation between the anterior lens capsule PXF material and IOP or the indices of glaucoma severity (P>0.42). The grade of angle pigmentation, but not lens PXF, in eyes with IOP >21 mm Hg was significantly higher than in eyes with IOP ≤21 mm Hg (P=0.04). ConclusionsIn patients with PXF syndrome/glaucoma, gonioscopically identified iridocorneal angle pigmentation correlates more strongly with presenting IOP than the amount of PXF material on the anterior lens capsule.

Synergistic activation of guinea‐pig cardiac cystic fibrosis transmembrane conductance regulator by the tyrosine kinase inhibitor genistein and cAMP

1 The regulation of cardiac Cl− current (IC1) by tyrosine and serine/threonine phosphorylation was examined in guinea‐pig and rat ventricular myocytes. The protein tyrosine kinase (PTK) inhibitor genistein (GST) and phosphotyrosine phosphatase (PTP) inhibitor sodium orthovanadate (VO4) were used to modify tyrosine phosphorylation, whereas forskolin (FSK), cAMP, and other agents were used to modify cytoplasmic cAMP concentration and protein kinase A (PKA) phosphorylation. 2 Low concentrations (0.1 μm) of FSK did not activate the PKA‐regulated cystic fibrosis transmembrane regulator (CFTR) ICl in guinea‐pig ventricular myocytes, but strongly potentiated activation of an ICl by 20–100 μm GST. The potentiation did not occur when GST was replaced by PTK‐inactive daidzein, and it was strongly inhibited by 1 mm VO4. 3 Potentiation by 0.1 μm FSK was linked to a small stimulation of the adenylate cyclase–cAMP–PKA pathway. The potentiation was not mimicked by inactive 1,9‐dideoxyforskolin, and was inhibited by muscarinic stimulation (ACh) and by a PKA inhibitor. Internal application of a cAMP solution that alone was too weak to activate CFTR ICl strongly potentiated the activation of ICl by 50 μm GST and occluded potentiation by 0.1 μm FSK. 4 The foregoing suggests that potentiated ICl flows through cAMP‐dependent CFTR channels. In agreement with this interpretation, GST did not increase ICl when CFTR was maximally activated by a high concentration (5 μm) of FSK and okadaic acid, and neither GST nor GST plus FSK activated an ICl in CFTR‐deficient rat myocytes. The lack of effect in rat myocytes was not due to the absence of functional, channel‐relevant PKA and PTK–PTP systems, because (as in guinea‐pig myocytes) L‐type Ca2+ current (ICa,L) was stimulated by FSK and inhibited in a VO4‐reversible manner by GST. 5 The synergistic activation of CFTR by low concentrations of FSK and GST cannot be explained by either a GST‐induced elevation of cAMP concentration or inhibition of serine/threonine phosphatase. Rather, it appears to be due to tyrosine dephosphorylation that facilitates PKA‐mediated phosphorylation of the channels.

Phorbol ester activation of chloride current in guinea‐pig ventricular myocytes

1 Although earlier studies with phorbol esters indicate that protein kinase C (PKC) may be an important regulator of Cl− current (ICl) in cardiac cells, there is a need for additional quantitative data and investigation of conflicting findings. Our objectives were to measure the magnitude, time course, and concentration‐dependence of ICl activated in guinea‐pig ventricular myocytes by phorbol 12‐myristate 13‐acetate (PMA), evaluate its PKC dependence, and examine its modification by external and internal ions. 2 The whole‐cell patch clamp technique was used to apply short depolarizing and hyperpolarizing pulses to myocytes superfused with Na+‐, K+‐, Ca2+‐free solution (36°C) and dialysed with Cs+ solution. Stimulation of membrane currents by PMA (threshold ≤1nM, EC5014nM, maximal 40% increase with ≥100 nM) plateaued within 6–10 min. 3 PMA‐activated current was time‐independent, and suppressed by 1 mM 9‐anthracenecarboxylic acid (9‐AC). Its reversal potential (Erev) was sensitive to changes in the Cl− gradient, and outward rectification of the current‐voltage (I‐V) relationship was more pronounced with 30 mM than 140 mM Cl− dialysate. 4 The relative permeability of PMA‐activated channels estimated from Erev measurements was I−>Cl− » aspartate. Channel activation was independent of external Na+. 5 PMA failed to activate ICl in myocytes pretreated with 1‐(5‐isoquinolinesulphonyl)‐2‐methylpiperazine (H‐7) or dialysed with pCa 10.5 solution. Lack of response to 4α‐phorbol 12, 13‐didecanoate (αPDD) was a further indication of mediation by PKC. 6 ICl induced by 2 μm forskolin was far larger than that induced by PMA, suggesting that endogenous protein kinase A is a much stronger Cl− channel activator than endogenous PKC in these myocytes. 7 The macroscopic properties of PMA‐induced ICl appear to be indistinguishable from those of PKA‐activated ICl. We discount stimulation of PKA by PMA as an explanation, and conclude that endogenous PKC may activate PKA‐regulated Cl− channels in these myocytes.

Beta and Gamma Peripapillary Atrophy in Myopic Eyes With and Without Glaucoma

PURPOSE To determine whether beta and gamma peripapillary atrophy (PPA) areas measured with optical coherence tomography (OCT) enhances glaucoma diagnosis in myopic subjects. METHODS We included 55 myopic glaucoma patients and 74 myopic nonglaucomatous controls. Beta-PPA comprised the area external to the clinical disc margin, with absence of retinal pigment epithelium and presence of Bruchs membrane. Gamma-PPA comprised the area external to the disc margin, with absence of both RPE and Bruchs membrane. OCT scans colocalized to fundus photographs were used to measure PPA, choroidal thickness, border tissue of Elschnig configuration, optic disc area, and optic disc ovality. RESULTS Beta-PPA area was larger in glaucoma patients compared with controls (median [interquartile range], 1.0 [0.66-1.53] mm2 and 0.74 [0.50-1.38] mm2, respectively), whereas gamma-PPA was smaller in glaucoma patients compared with controls (0.28 [0.14-0.50] mm2 and 0.42 [0.17-0.74] mm2, respectively). However, the distributions of both beta- and gamma-PPA in the two groups overlapped widely. The areas under the receiver operating characteristic curve of beta- and gamma-PPA areas were 0.60 and 0.59, respectively. Larger beta-PPA area was associated with larger disc area, thinner choroidal thickness, longer axial length, less oblique border tissue configuration, older age, and greater disc ovality. Larger gamma-PPA area was associated with greater disc ovality, more oblique border tissue configuration, and longer axial length. CONCLUSIONS Subclassifying PPA with OCT into beta and gamma zones reveals association with different covariates, but does not enhance the diagnostic performance for glaucoma in a population of predominantly Caucasians myopic subjects.