Kimberly A. Mankiewicz

Allosteric mechanism in AMPA receptors: A FRET-based investigation of conformational changes

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the primary mediators of fast excitatory synaptic transmission in the mammalian CNS. Structures of the extracellular ligand-binding domain suggest that the extent of cleft closure in the ligand-binding domain controls the extent of activation of the receptor. Here we have developed a fluorescence resonance energy transfer-based probe that allows us to study the extent of cleft closure in the isolated ligand-binding domain in solution. These investigations show that the wild-type protein exhibits a graded cleft closure that correlates to the extent of activation, which is in qualitative agreement with the crystal structures. However, the changes in extent of cleft closure between the apo and agonist-bound states are smaller than that observed in the crystal structures. We have also used this method to study the L650T mutant and show that in solution the α-amino-5-methyl-3-hydroxy-4-isoxazole propionate-bound form of this mutant exists primarily in a conformation that is more closed than predicted based on the activity, indicating that the degree of cleft closure alone cannot be used as a measure of extent of activation of the receptor, and there are possibly other mechanisms in addition to cleft closure that mediate the subtleties in extent of activation by a given agonist.

Aqueous shunt exposure: a retrospective review of repair outcome.

PurposeTo describe the patient outcomes and factors affecting those outcomes after aqueous shunt exposure repair. Patients and MethodsForty-three eyes from Robert Cizik Eye Clinic and Bascom Palmer Eye Institute from 1995 to 2007 suffered from aqueous shunt exposure and were repaired by participating surgeons. Thirty-three were tube exposures and 7 were plate exposures. The remaining 3 exposure classified complications included a patch graft exposure, an elbow exposure, and 1 unknown complication. Forty eyes were followed for evidence of additional aqueous shunt exposures or additional surgical interventions for 46.6 weeks (40.2 wk) (range: 3 to 168 wk). ResultsSeventeen of 40 eyes required additional surgical intervention: 15 (45%) from the tube exposure group and 2 (29%) from the plate exposure group. Five (13%) eyes needed eventual removal of the shunt. Black race, diabetes mellitus, a high number of glaucoma medications before shunt implantation, a history of multiple glaucoma laser procedures, and combination of an initial aqueous shunt implantation with another surgery were found to be associated with a worse outcome after exposure repair. ConclusionsIntraocular pressure, number of medications, and visual acuity remained stable during follow-up after revision. Diabetes mellitus was associated with a shorter average time between initial repair and reintervention, and 4 other variables were associated with a higher likelihood of reintervention.

Reproducibility of Scleral Spur Identification and Angle Measurements Using Fourier Domain Anterior Segment Optical Coherence Tomography

Purpose. To evaluate intraobserver and interobserver agreement in locating the scleral spur landmark (SSL) and anterior chamber angle measurements obtained using Fourier Domain Anterior Segment Optical Coherence Tomography (ASOCT) images. Methods. Two independent, masked observers (SR and AZC) identified SSLs on ASOCT images from 31 eyes with open and nonopen angles. A third independent reader, NPB, adjudicated SSL placement if identifications differed by more than 80 μm. Nine months later, SR reidentified SSLs. Intraobserver and interobserver agreement in SSL placement, trabecular-iris space area (TISA750), and angle opening distance (AOD750) were calculated. Results. In 84% of quadrants, SRs SSL placements during 2 sessions were within 80 μm in both the X- and Y-axes, and in 77% of quadrants, SR and AZC were within 80 μm in both axes. In adjudicated images, 90% of all quadrants were within 80 μm, 88% in nonopen-angle eyes, and 92% in open-angle eyes. The intraobserver and interobserver correlation coefficients (with and without adjudication) were above 0.9 for TISA750 and AOD750 for all quadrants. Conclusions. Reproducible identification of the SSL from images obtained with FD-ASOCT is possible. The ability to identify the SSL allows reproducible measurement of the anterior chamber angle using TISA750 and AOD750.

Trabecular-Iris Circumference Volume in Open Angle Eyes Using Swept-Source Fourier Domain Anterior Segment Optical Coherence Tomography

Purpose. To introduce a new anterior segment optical coherence tomography parameter, trabecular-iris circumference volume (TICV), which measures the integrated volume of the peripheral angle, and establish a reference range in normal, open angle eyes. Methods. One eye of each participant with open angles and a normal anterior segment was imaged using 3D mode by the CASIA SS-1000 (Tomey, Nagoya, Japan). Trabecular-iris space area (TISA) and TICV at 500 and 750 µm were calculated. Analysis of covariance was performed to examine the effect of age and its interaction with spherical equivalent. Results. The study included 100 participants with a mean age of 50 (±15) years (range 20–79). TICV showed a normal distribution with a mean (±SD) value of 4.75 µL (±2.30) for TICV500 and a mean (±SD) value of 8.90 µL (±3.88) for TICV750. Overall, TICV showed an age-related reduction (P = 0.035). In addition, angle volume increased with increased myopia for all age groups, except for those older than 65 years. Conclusions. This study introduces a new parameter to measure peripheral angle volume, TICV, with age-adjusted normal ranges for open angle eyes. Further investigation is warranted to determine the clinical utility of this new parameter.

Optimal number of angle images for calculating anterior angle volume and iris volume measurements

PURPOSE We determined the optimal number of angle images required to obtain reliable measurements of trabecular-iris circumferential volume (TICV) and iris volume (IV) using swept-source Fourier domain anterior segment optical coherence tomography (SSFD-ASOCT) scans in narrow angle eyes. METHODS Scleral spur landmarks (SSL) were manually identified on ASOCT angle images from 128 meridians from each of 24 eyes with chronic primary angle closure (PAC) spectrum of disease. The anterior and posterior corneal curves, and the anterior and posterior iris surfaces were identified automatically by the anterior chamber analysis and interpretation (ACAI) software, then manually examined and edited by the reader if required. Trabecular-iris circumferential volume at 750 μm from SSL (TICV750) and IV were subsequently calculated using varying numbers of angle images. Threshold error was determined to be less than the lower 95% confidence limit of mean absolute percent error (MAPE) of the change in TICV or IV resulting from laser peripheral iridotomy, which would be 17% for TICV and 5% for IV, based on previous studies. The optimal number of angle images was the smallest number of images where MAPE was less than this threshold for TICV and IV. RESULTS A total of 32 equally-spaced angle images (16 meridians) was required to estimate TICV750 and 16 angle images (8 meridians) to estimate IV. Both were within 4.6% and 1.6% of MAPE, respectively. CONCLUSIONS It is possible to determine TICV and IV parameters reliably in narrow angles without evaluating all 128 meridians obtained with SSFD-ASOCT.

Intermediate term safety and efficacy of transscleral cyclophotocoagulation after tube shunt failure

PurposeTo determine the efficacy and safety of diode transscleral cyclophotocoagulation (TSCPC) after tube shunt failure. Patients and MethodsThe patient population consisted of 32 eyes of 31 patients with uncontrolled glaucoma. Each eye had a previously implanted aqueous tube shunt and was currently on maximally tolerated medication. Each eye also underwent TSCPC treatment using the Iridex (Mountain View, CA) diode laser with a maximum of 360 degrees of treatment. All 31 charts were reviewed for data pertaining to demographics, treatment, ocular history, and follow-up clinical examinations. Safety was evaluated by complication data. Efficacy was evaluated in terms of TSCPC treatment parameters (number of laser applications, laser power, application duration, and degrees of ciliary body treated), intraocular pressure, number of hypotensive medications, and any further treatment required. ResultsWith a mean (SD) follow-up of 17.1 (16.3) (median=11.7) months from the last treatment, the mean intraocular pressure decreased from 28.6 (10.2) mm Hg to 16.8 (7.5) mm Hg (35% reduction) at 3 months (n=30, P<0.0001) and to 14.7 (7.9) mm Hg (43% reduction) at 1 year (n=13, P<0.0001). Complications included hypotony (n=4), hyphema (n=2), failed corneal transplant (n=1), and loss of light perception (n=5). ConclusionsTSCPC has a significant ocular hypotensive effect on glaucoma refractory to both tube shunt and medical therapy. The safety of this intervention remains unclear in this high risk patient population and warrants further study.

Effect of Laser Peripheral Iridotomy on Anterior Chamber Angle Anatomy in Primary Angle Closure Spectrum Eyes.

Purpose:To evaluate the change in trabecular-iris circumference volume (TICV) after laser peripheral iridotomy (LPI) in primary angle closure (PAC) spectrum eyes. Patients and Methods:Forty-two chronic PAC spectrum eyes from 24 patients were enrolled. Eyes with anterior chamber abnormalities affecting angle measurement were excluded. Intraocular pressure, slit lamp examination, and gonioscopy were recorded at each visit. Anterior segment optical coherence tomography (ASOCT) with 3D mode angle analysis scans were taken with the CASIA SS-1000 before and after LPI. Forty-two pre-LPI ASOCT scans and 34 post-LPI ASOCT scans were analyzed using the Anterior Chamber Analysis and Interpretation (ACAI) software. A mixed-effect model analysis was used to compare the trabecular-iris space area (TISA) changes among 4 quadrants, as well as to identify potential factors affecting TICV. Results:There was a significant increase in all average angle parameters after LPI (TISA500, TISA750, TICV500, and TICV750). The magnitude of change in TISA500 in the superior angle was significantly less than the other angles. The changes in TICV500 and TICV750 were not associated with any demographic or ocular characteristics. Conclusions:TICV is a useful parameter to quantitatively measure the effectiveness of LPI in the treatment of eyes with PAC spectrum disease.

Age-Related Changes in Trabecular Meshwork Imaging

Purpose. To evaluate the normal aging effects on trabecular meshwork (TM) parameters using Fourier domain anterior segment optical coherence tomography (ASOCT) images. Patients and Methods. One eye from 45 participants with open angles was imaged. Two independent readers measured TM area, TM length, and area and length of the TM interface shadow from 3 age groups (18–40, 41–60, and 61–80). Measurements were compared using stepwise regression analysis. Results. The average TM parameters were 0.0487 (±0.0092) mm2 for TM area, 0.5502 (±0.1033) mm for TM length, 0.1623 (±0.341) mm2 for TM interface shadow area, and 0.7755 (±0.1574) mm for TM interface shadow length. Interobserver reproducibility coefficients ranged from 0.45 (TM length) to 0.82 (TM area). TM area and length were not correlated with age. While the TM interface shadow length did not correlate with age, the TM interface shadow area increased with age. Race, sex, intraocular pressure, and gonioscopy score were not correlated with any TM parameters. Conclusion. Although the TM measurements were not correlated with age, the TM interface shadow area increased with age. Further study is required to determine whether there is any relationship between the age-related ASOCT findings of the TM interface shadow area and physiologic function.

Clinical Outcomes of Peripheral Iridotomy in Patients with the Spectrum of Chronic Primary Angle Closure

Purpose. To evaluate outcomes of peripheral iridotomy (PI) for initial management of primary angle closure suspects (PACS), chronic primary angle closure (CPAC), and chronic primary angle closure glaucoma (CPACG). Patients and Methods. Seventy-nine eyes with PACS, CPAC, or CPACG and better than 20/50 visual acuity that underwent PI as initial management were included. Eyes with previous acute angle closure attacks, laser trabeculoplasties, surgeries, or intraocular injections were excluded. Additional treatments, glaucomatous progression, intraocular pressure, visual acuity, and the number of medications were evaluated. Results. The mean followup was 57.1 ± 29.0 months (range 13.8–150.6 months). Sixty-eight eyes (86.1%) underwent additional medical, laser, or surgical treatment. Forty eyes (50.6%) underwent lens extraction due to reduced visual acuity. The mean 10× logMAR visual acuity score for all patients significantly declined from 0.94 ± 1.12 at baseline to 1.83 ± 3.49 (N = 79, P = 0.0261) at the last followup. Conclusions. Most patients who undergo PI for CPAC spectrum will require additional intervention for either IOP lowering or improvement of visual acuity. This suggests that a procedure that not only deepens the angle but also lowers IOP and improves visual acuity would be desirable as further intervention could be avoided. Evaluation of techniques that achieve all 3 goals is warranted.

Glutamate receptors as seen by light: spectroscopic studies of structure-function relationships

Ionotropic glutamate receptors are major excitatory receptors in the central nervous system and also have a far reaching influence in other areas of the body. Their modular nature has allowed for the isolation of the ligand-binding domain and for subsequent structural studies using a variety of spectroscopic techniques. This review will discuss the role of specific ligand:protein interactions in mediating activation in the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of glutamate receptors as established by various spectroscopic investigations of the GluR2 and GluR4 subunits of this receptor. Specifically, this review will provide an introduction to the insight gained from X-ray crystallography and nuclear magnetic resonance investigations and then go on to focus on studies utilizing vibrational spectroscopy and fluorescence resonance energy transfer to study the behavior of the isolated ligand-binding domain in solution and discuss the importance of specific ligand:protein interactions in the mechanism of receptor activation.