Amir H. Kashani

Calcium Regulation of Dendritic Growth via CaM Kinase IV and CREB-Mediated Transcription

We report that CaM kinase IV and CREB play a critical role in mediating calcium-induced dendritic growth in cortical neurons. Calcium-dependent dendritic growth is suppressed by CaM kinase inhibitors, a constitutively active form of CaM kinase IV induces dendritic growth in the absence of extracellular stimulation, and a kinase-dead form of CaM kinase IV suppresses dendritic growth induced by calcium influx. CaM kinase IV activates the transcription factor CREB, and expression of a dominant negative form of CREB blocks calcium- and CaM kinase IV-induced dendritic growth. In cortical slice cultures, dendritic growth is attenuated by inhibitors of voltage-sensitive calcium channels and by dominant negative CREB. These experiments indicate that calcium-induced dendritic growth is regulated by activation of a transcriptional program that involves CaM kinase IV and CREB-mediated signaling to the nucleus.

OCT angiography in healthy human subjects.

BACKGROUND AND OBJECTIVE To noninvasively evaluate the retinal microvasculature in healthy human subjects with optical coherence tomography angiography (OCTA). PATIENTS AND METHODS Cross-sectional, observational study of five healthy subjects. OCTA was performed on 3 × 3 mm(2) sections centered on the fovea, nasal macula, and temporal macula. Retinal vasculature was assessed within three horizontal slabs consisting of the inner, middle, and outer retina. The vasculature within each retinal slab was reconstructed using phase-based and intensity contrast-based algorithms and visualized as separate en face images. RESULTS OCTA in healthy subjects demonstrates capillary networks consistent with previous histological studies. No retinal vessels were found in the outer retina. OCT angiography of the inner and middle retinal layers showed region-specific vascular patterns that consistently corroborated qualitative findings from past histological studies. CONCLUSION OCTA generates high-resolution, noninvasive angiograms qualitatively similar to conventional fluorescein angiography. OCTA may serve as a bridge to assess some features of the retinal microvasculature between conventionally performed angiograms.

Retinal Thickness Analysis by Race, Gender, and Age Using Stratus OCT

PURPOSE To detect differences in retinal thickness among patients of different race, gender, and age using Stratus OCT. DESIGN Cross-sectional study. METHODS In a multicenter, university-based study, 126 patients with no history of ocular disease were enrolled (78 diabetics without retinopathy and 48 nondiabetics). Optical coherence tomography measurements were performed using Stratus OCT. Statistical comparisons of center point foveal thickness and mean foveal thickness were made using generalized estimating equations adjusting for diabetic status, race, age, and gender. RESULTS The study population consisted of 36% male subjects, 39% Caucasian, 33% African-American, and 28% Hispanic. Mean foveal thickness was 191.6 +/- 2.7 microm and 194.5 +/- 2.7 microm for diabetics and nondiabetics, respectively (P = .49). Mean foveal thickness in male subjects was significantly larger than in female (201.8 +/- 2.7 microm and 186.9 +/- 2.6 microm, respectively; P < .001). Mean foveal thickness was 200.2 +/- 2.7 microm for Caucasian, 181.0 +/- 3.7 microm for African-American, and 194.7 +/- 3.9 microm for Hispanic subjects. Mean foveal thickness was significantly less for African-American than Caucasian (P < .0001) or Hispanic subjects (P = .005). Center point foveal thickness and mean foveal thickness showed a significant increase with age. CONCLUSIONS There are statistically significant differences in retinal thickness between subjects of different race, gender, and age. When compared to Caucasian and Hispanic subjects, African-American race is a predictor of decreased mean foveal thickness; and male sex (regardless of race) is a significant predictor of increased mean foveal thickness. Mean foveal thickness is similar among diabetics and nondiabetics when data are controlled for age, race, and sex. These results suggest that studies comparing OCT measurements should carefully control for age-based, race-based, and gender-based variations in retinal thickness.

Quantifying Microvascular Density and Morphology in Diabetic Retinopathy Using Spectral-Domain Optical Coherence Tomography Angiography.

Purpose To quantify changes in retinal microvasculature in diabetic retinopathy (DR) by using spectral-domain optical coherence tomography angiography (SD-OCTA). Methods Retrospective, cross-sectional, observational study of healthy and diabetic adult subjects with and without DR. Retinal microvascular changes were assessed by using SD-OCTA images and an intensity-based optical microangiography algorithm. A semiautomated program was used to calculate indices of microvascular density and morphology in nonsegmented and segmented SD-OCTA images. Microvascular density was quantified by using skeleton density (SD) and vessel density (VD), while vessel morphology was quantified as fractal dimension (FD) and vessel diameter index (VDI). Statistical analyses were performed by using the Students t-test or analysis of variance with post hoc Tukey honest significant difference tests for multiple comparisons. Results Eighty-four eyes with DR and 14 healthy eyes were studied. Spearmans rank test demonstrated a negative correlation between DR severity and SD, VD, and FD, and a positive correlation with VDI (ρ = −0.767, −0.7166, −0.768, and +0.5051, respectively; P < 0.0001). All parameters showed high reproducibility between graders (ICC = 0.971, 0.962, 0.937, and 0.994 for SD, VD, FD, and VDI, respectively). Repeatability (κ) was greater than 0.99 for SD, VD, FD, and VDI. Conclusions Vascular changes in DR can be objectively and reliably characterized with SD, VD, FD, and VDI. In general, decreasing capillary density (SD and VD), branching complexity (FD), and increasing average vascular caliber (VDI) were associated with worsening DR. Changes in capillary density and morphology were significantly correlated with diabetic macular edema.

Calcium activation of the LMO4 transcription complex and its role in the patterning of thalamocortical connections

Lasting changes in neuronal connectivity require calcium-dependent gene expression. Here we report the identification of LIM domain-only 4 (LMO4) as a mediator of calcium-dependent transcription in cortical neurons. Calcium influx via voltage-sensitive calcium channels and NMDA receptors contributes to synaptically induced LMO4-mediated transactivation. LMO4-mediated transcription is dependent on signaling via calcium/calmodulin-dependent protein (CaM) kinase IV and microtubule-associated protein (MAP) kinase downstream of synaptic stimulation. Coimmunoprecipitation experiments indicate that LMO4 can form a complex with cAMP response element-binding protein (CREB) and can interact with cofactor of LIM homeodomain protein 1 (CLIM1) and CLIM2. To evaluate the role of LMO4 in vivo, we examined the consequences of conditional loss of lmo4 in the forebrain, using the Cre-Lox gene-targeting strategy. The organization of the barrel field in somatosensory cortex is disrupted in mice in which lmo4 is deleted conditionally in the cortex. Specifically, in contrast to controls, thalamocortical afferents in conditional lmo4 null mice fail to segregate into distinct barrel-specific domains. These observations identify LMO4 as a calcium-dependent transactivator that plays a key role in patterning thalamocortical connections during development.

Stem cell based therapies for age-related macular degeneration: The promises and the challenges

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in developed countries. AMD is classified as either neovascular (NV-AMD) or non-neovascular (NNV-AMD). Cumulative damage to the retinal pigment epithelium, Bruchs membrane, and choriocapillaris leads to dysfunction and loss of RPE cells. This causes degeneration of the overlying photoreceptors and consequential vision loss in advanced NNV-AMD (Geographic Atrophy). In NV-AMD, abnormal growth of capillaries under the retina and RPE, which leads to hemorrhage and fluid leakage, is the main cause of photoreceptor damage. Although a number of drugs (e.g., anti-VEGF) are in use for NV-AMD, there is currently no treatment for advanced NNV-AMD. However, replacing dead or dysfunctional RPE with healthy RPE has been shown to rescue dying photoreceptors and improve vision in animal models of retinal degeneration and possibly in AMD patients. Differentiation of RPE from human embryonic stem cells (hESC-RPE) and from induced pluripotent stem cells (iPSC-RPE) has created a potentially unlimited source for replacing dead or dying RPE. Such cells have been shown to incorporate into the degenerating retina and result in anatomic and functional improvement. However, major ethical, regulatory, safety, and technical challenges have yet to be overcome before stem cell-based therapies can be used in standard treatments. This review outlines the current knowledge surrounding the application of hESC-RPE and iPSC-RPE in AMD. Following an introduction on the pathogenesis and available treatments of AMD, methods to generate stem cell-derived RPE, immune reaction against such cells, and approaches to deliver desired cells into the eye will be explored along with broader issues of efficacy and safety. Lastly, strategies to improve these stem cell-based treatments will be discussed.

Optical Coherence Tomography Angiography of Diabetic Retinopathy in Human Subjects

BACKGROUND AND OBJECTIVE Optical coherence tomography angiography (OCTA) is a novel, non-invasive OCT technique capable of imaging the retinal vasculature. This study aims to evaluate the retinal microvasculature in diabetic human subjects with OCTA and assess potential clinical applications. PATIENTS AND METHODS Cross-sectional study of 33 subjects with diabetic retinopathy. OCTA was performed on 3 mm × 3 mm sections using a swept-source OCTA prototype and a phase- and intensity-based contrasting algorithm. OCT angiograms were studied with corresponding clinical examination and fluorescein angiograms, when available, to assess accuracy and clinical utility. RESULTS OCTA was able to demonstrate most clinically relevant vascular changes in subjects with diabetic retinopathy, including microaneurysms, impaired vascular perfusion, some forms of intraretinal fluid, vascular loops, intraretinal microvascular abnormalities, neovascularization, and cotton-wool spots that were largely consistent with fluorescein angiography. CONCLUSION OCTA generates high-resolution angiograms that illustrate many of the clinically relevant findings in diabetic retinopathy and offers a novel complement or alternative to fluorescein angiography. Although currently an investigational technique, OCTA in combination with standard OCT imaging is at least as good as fluorescein angiography in the evaluation of the macular complications of diabetic retinopathy.

Hypertonic enhancement of transmitter release from frog motor nerve terminals: Ca2+ independence and role of integrins

1 Hyperosmotic solutions cause markedly enhanced spontaneous quantal release of neurotransmitter from many nerve terminals. The mechanism of this enhancement is unknown. We have investigated this phenomenon at the frog neuromuscular junction with the aim of determining the degree to which it resembles the modulation of release by stretch, which has been shown to be mediated by mechanical tension on integrins. 2 The hypertonicity enhancement, like the stretch effect, does not require Ca2+ influx or release from internal stores, although internal release may contribute to the effect. 3 The hypertonicity effect is sharply reduced (but not eliminated) by peptides containing the RGD sequence, which compete with native ligands for integrin bonds. 4 There is co‐variance in the magnitude of the stretch and osmotic effects; that is, individual terminals exhibiting a large stretch effect also show strong enhancement by hypertonicity, and vice versa. The stretch and osmotic enhancements also can partially occlude each other. 5 There remain some clear‐cut differences between osmotic and stretch forms of modulation: the larger range of enhancement by hypertonic solutions, the relative lack of effect of osmolarity on evoked release, and the reported higher temperature sensitivity of osmotic enhancement. Nevertheless, our data strongly implicate integrins in a significant fraction of the osmotic enhancement, possibly acting via the same mechanism as stretch modulation.

OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY OF RETINAL VENOUS OCCLUSION.

Purpose: To noninvasively evaluate the retinal microvasculature in human subjects with retinal venous occlusions using optical coherence tomography angiography and assess potential clinical applications. Methods: This was a prospective, observational study of adult human subjects with clinical and imaging findings demonstrating retinal venous occlusion. Subjects underwent complete ophthalmic examination and fluorescein angiography as appropriate for their standard of care. Optical coherence tomography angiography was performed on a prototype spectral domain-OCTA system in 3 mm × 3 mm and 6 mm × 6 mm regions centered on the fovea and parafoveal areas. Retinal vasculature was assessed within three horizontal slabs consisting of the superficial, middle, and deep retina. The vasculature within each slab was reconstructed using intensity contrast-based algorithms and visualized as en-face images. Optical coherence tomography angiograms were manually segmented to verify the accuracy of the automated segmentation algorithms. Results: Optical coherence tomography angiography was able to demonstrate almost all of the clinically relevant findings in 25 subjects with acute and chronic retinal venous occlusion. These findings were consistent with clinical, anatomic, and fluorescein angiographic findings including areas of impaired vascular perfusion, retinal atrophy, vascular dilation, shunt vessels, and some forms of intraretinal edema. Conclusion: Optical coherence tomography angiography is an investigational method that generates high-resolution, noninvasive angiograms that qualitatively illustrate most of clinically relevant findings in retinal venous occlusion. Optical coherence tomography angiography corresponds well with fluorescein angiograms and in many cases provides more detailed anatomic and blood flow information. Optical coherence tomography angiography, in conjunction with standard spectral domain-OCT, is at least equally as effective as fluorescein angiography for evaluation and management of the macular complications of retinal venous occlusions.

Quantitative Subanalysis of Cystoid Spaces and Outer Nuclear Layer Using Optical Coherence Tomography in Age-Related Macular Degeneration

PURPOSE To use optical coherence tomography (OCT) to quantify intraretinal cystoid spaces (ICSs) and the outer nuclear layer (ONL) in patients with neovascular age-related macular degeneration (AMD) and to investigate the correlation of these parameters with visual acuity. METHODS StratusOCT (Carl Zeiss Meditec, Inc., Dublin, CA) images were collected from 53 patients receiving their initial treatment with intravitreous ranibizumab. Images were analyzed with custom software (OCTOR) that allows accurate manual segmentation of OCT B-scans and provides thickness/volume measurements of ICS, ONL, neurosensory retina, pigment epithelial detachments (PEDs), subretinal fluid (SRF), and subretinal tissue (SRT). Univariate and multivariate analyses were used to correlate OCT parameters with best corrected Snellen visual acuity. Reproducibility was assessed with weighted kappa statistics and intraclass correlation coefficients. RESULTS A multivariate linear regression model with adjusted R(2) showed that ONL volume and SRT thickness significantly correlated with Snellen visual acuity (R(2) = 0.15, P = 0.002 and R(2) = 0.19, P = 0.001, respectively) with an overall model R(2) of 0.34. Adjustment of ONL volume for ICS did not improve correlation with visual acuity, and ICS volume did not independently correlate with visual acuity. Weighted kappa statistics showed excellent intergrader agreement for both ICS and ONL measurements. CONCLUSIONS The results suggest that an increased total volume of the ONL is associated with decreased visual acuity in neovascular AMD and that the total volume of ICS does not correlate with visual acuity. Although the correlations detected in this study are modest, quantitative subanalysis of OCT images may be of greater clinical relevance in the context of more advanced OCT technology.