Paul J. Mackenzie

Ca2+ Imaging of CNS Axons in Culture Indicates Reliable Coupling between Single Action Potentials and Distal Functional Release Sites

A combination of Ca2+ imaging and current clamp recording in cultured cortical neurons was used to evaluate the reliability of coupling between the action potential and rises in Ca2+ at distal release sites as a possible source of variability in CNS synaptic transmission. Local domains of enhanced Ca2+ influx were observed at varicosities on axon collaterals. Functional assay of vesicle turnover using FM1-43 and parallel electron microscopy confirmed that these varicosities were release sites. Single action potentials reliably ( > 95% of the time) resulted in a presynaptic Ca2+ transient at all presumed release sites including those on distal collaterals. Variability in the amplitude of presynaptic Ca2+ transients at individual boutons was estimated to be on average less than 20%. We conclude that the coupling of somatic action potentials to distal release sites is generally a reliable process, although nonlinearity in the relationship between Ca2+ influx and neurotransmitter release may amplify the effects of relatively small fluctuations in Ca2+ influx.

Sensitivity and specificity of the optos optomap for detecting peripheral retinal lesions.

Purpose: To compare the sensitivity and specificity of the Optomap Panoramic200 wide-field confocal scanning laser imaging system for detecting peripheral retinal lesions. Methods: Optomap images were obtained in patients with known retinal pathology. Two masked retinal specialists evaluated Optomap images to identify lesions requiring referral to a retinal specialist. Their performance was compared to gold standard examination with scleral indentation performed by a retinal specialist. Sensitivity was calculated overall and again for lesions that were found on clinical examination to require treatment. These sensitivities were calculated separately for lesions posterior and anterior to the equator. Specificity was calculated from fellow eyes that were found to have no pathology on clinical examination. Results: For retinal lesions posterior to the equator, sensitivity was 74% (95% confidence interval [95% CI] 61%–87%) overall for all lesions and 76% (95% CI 59%–93%) for lesions requiring treatment. For anterior lesions, sensitivity was 45% (95% CI 28%–62%) overall and 36% (95% CI 14%–58%) for treatable lesions. Specificity was 85% (95% CI 63%–100%). Conclusions: The Optomap showed high specificity and moderate sensitivity for lesions posterior to the equator and low sensitivity for lesions anterior to the equator.

Vascular anatomy of the optic nerve head

Although intraocular pressure remains the main modifiable risk factor for open-angle glaucoma, other factors such as vascular perfusion likely play a significant role. It is not clear how mechanical deformation, axonal damage, glial responses, and ischemia interact to lead to the tissue remodeling seen clinically as glaucomatous cupping. To understand the potential role of vascular risk factors in glaucoma, it is important to understand the vascular anatomy of the optic nerve head (ONH). The focus of this review is to provide a description of the vascular anatomy of the ONH and to describe recent work in the central nervous system that suggests that astrocytes play a key role in vascular regulation. Finally, the evidence for vascular regulation in the ONH and retina is reviewed.

Rapid volumetric OCT image acquisition using compressive sampling.

Acquiring three dimensional image volumes with techniques such as Optical Coherence Tomography (OCT) relies on reconstructing the tissue layers based on reflection of light from tissue interfaces. One B-mode scan in an image is acquired by scanning and concatenating several A-mode scans, and several contiguous slices are acquired to assemble a full 3D image volume. In this work, we demonstrate how Compressive Sampling (CS) can be used to accurately reconstruct 3D OCT images with minimal quality degradation from a subset of the original image. The full 3D image is reconstructed from sparsely sampled data by exploiting the sparsity of the image in a carefully chosen transform domain. We use several sub-sampling schemes, recover the full 3D image using CS, and show that there is negligible effect on clinically relevant morphometric measurements of the optic nerve head in the recovered image. The potential outcome of this work is a significant reduction in OCT image acquisition time, with possible extensions to speeding up acquisition in other imaging modalities such as ultrasound and MRI.

Real-time high-speed volumetric imaging using compressive sampling optical coherence tomography

Volumetric imaging of the Optic Nerve Head (ONH) morphometry with Optical Coherence Tomography (OCT) requires dense sampling and relatively long acquisition times. Compressive Sampling (CS) is an emerging technique to reduce volume acquisition time with minimal image degradation by sparsely sampling the object and reconstructing the missing data in software. In this report, we demonstrated real-time CS-OCT for volumetric imaging of the ONH using a 1060nm Swept-Source OCT prototype. We also showed that registration and averaging of CS-recovered volumes enhanced visualization of deep structures of the sclera and lamina cribrosa. This work validates CS-OCT as a means for reducing volume acquisition time and for preserving high-resolution in volume-averaged images. Compressive sampling can be integrated into new and existing OCT systems without changes to the optics, requiring only software changes and post-processing of acquired data.

Label-Free Density Measurements of Radial Peripapillary Capillaries in the Human Retina

Radial peripapillary capillaries (RPCs) comprise a unique network of capillary beds within the retinal nerve fibre layer (RNFL) and play a critical role in satisfying the nutritional requirements of retinal ganglion cell (RGC) axons. Understanding the topographical and morphological characteristics of these networks through in vivo techniques may improve our understanding about the role of RPCs in RGC axonal health and disease. This study utilizes a novel, non-invasive and label-free optical imaging technique, speckle variance optical coherence tomography (svOCT), for quantitatively studying RPC networks in the human retina. Six different retinal eccentricities from 16 healthy eyes were imaged using svOCT. The same eccentricities were histologically imaged in 9 healthy donor eyes with a confocal scanning laser microscope. Donor eyes were subject to perfusion-based labeling techniques prior to retinal dissection, flat mounting and visualization with the microscope. Capillary density and diameter measurements from each eccentricity in svOCT and histological images were compared. Data from svOCT images were also analysed to determine if there was a correlation between RNFL thickness and RPC density. The results are as follows: (1) The morphological characteristics of RPC networks on svOCT images are comparable to histological images; (2) With the exception of the nasal peripapillary region, there were no significant differences in RPC density measurements between svOCT and histological images; (3) Capillary diameter measurements were significantly greater in svOCT images compared to histology; (4) There is a positive correlation between RPC density and RNFL thickness. The findings in this study suggest that svOCT is a reliable modality for analyzing RPC networks in the human retina. It may therefore be a valuable tool for aiding our understanding about vasculogenic mechanisms that are involved in RGC axonopathies. Further work is required to explore the reason for some of the quantitative differences between svOCT and histology.

Quantitative Noninvasive Angiography of the Fovea Centralis Using Speckle Variance Optical Coherence Tomography

PURPOSE To demonstrate the utility of speckle variance optical coherence tomography (svOCT), a noninvasive angiographic technique, for evaluating the foveal vasculature. METHODS Twelve normal human eyes were imaged with svOCT (1060-nm, 100-kHz custom-built system) and fluorescein angiography (FA; Topcon TRC-50DX with 5.0 megapixel resolution camera). Manual tracing techniques were used to quantify the foveal vasculature, including foveal avascular zone (FAZ) metrics (area, perimeter, greatest diameter, and lowest diameter). Reproducibility of these measurements was determined. The FAZ was imaged in 25 normal eyes using svOCT and 15 donor eyes using confocal scanning laser microscopy. Retinal capillary plexuses in donor eyes were perfusion-labeled with phalloidin conjugated to Alexa Fluor 546. RESULTS Speckle variance OCT is able to stratify the foveal circulation into inner and deep capillary plexuses as well as reliably quantify and assess the morphometric dimensions of the human FAZ. Capillary density measurements were significantly greater in svOCT than FA (31.2 ± 1.6% vs. 19.3 ± 1.9% of total tissue area; P < 0.001). Measurements were highly reproducible (all P > 0.366). All FAZ metrics were significantly lower in histology than svOCT (all P < 0.001). CONCLUSIONS Speckle variance OCT permits precise, reproducible, and noninvasive visualization of the human foveal vasculature. Speckle variance OCT may become an important adjunct in evaluating patients with retinal vascular diseases.

In vivo optical imaging of human retinal capillary networks using speckle variance optical coherence tomography with quantitative clinico-histological correlation

Retinal capillary networks are critically linked to neuronal health and disease. The ability to perform accurate in vivo examination of human retinal capillary networks is therefore valuable for studying mechanisms that govern retinal homeostasis and retinal vascular diseases. Speckle variance optical coherence tomography (svOCT) is a non-invasive imaging technique that has the capacity to provide angiographic information about the retinal circulation. The application of this technology for studying human retinal capillary networks however has not been validated in a quantifiable manner. We use a custom-built svOCT device to qualitatively and quantitatively study the various capillary networks in the human perifovea. Capillary networks corresponding to the nerve fibre layer (NFL), the retinal ganglion cell/superficial inner plexiform layer (RGC/sIPL), the deep inner plexiform layer/superficial inner nuclear layer (dIPL/sINL) and the deep inner nuclear layer (dINL) are imaged in 9 normal human subjects. Measurements of capillary diameter and capillary density are made from each of these networks and results are compared to post-mortem histological data acquired with confocal scanning laser microscopy. Additionally, retinal capillary measurements from high-resolution fundus fluorescein angiogram (FA) are directly compared with svOCT images from 6 eyes. We demonstrate that svOCT images of capillary networks are morphologically comparable to microscopic images of histological specimens. Similar to histological images in svOCT images, the capillaries in the NFL network run parallel to the direction of RGC axons while capillaries in the dINL network comprise a planar configuration with multiple closed loops. Capillaries in remaining networks are convoluted with a complex three-dimensional architecture. We demonstrate that there is no significant difference in capillary density measurements between svOCT and histology images for all networks. Capillary diameter was significantly greater in svOCT images compared to histology for all networks. Capillary density measurements were also higher in svOCT compared to FA. The results of this study suggest that in vivo svOCT imaging allows accurate morphometric assessment of capillary networks in the human perifovea and may provide an improved ability to render microvascular detail compared to FA. Therefore, svOCT may have broad clinical applications in the study of human retinal physiology and disease. The difference in quantitative measurements between svOCT and histology may reflect dynamic variations in the retinal microcirculation and warrants further investigation.

Retinal angiography with real-time speckle variance optical coherence tomography

This report describes a novel, non-invasive and label-free optical imaging technique, speckle variance optical coherence tomography (svOCT), for visualising blood flow within human retinal capillary networks. This imaging system uses a custom-built swept source OCT system operating at a line rate of 100 kHz. Real-time processing and visualisation is implemented on a consumer grade graphics processing unit. To investigate the quality of microvascular detail acquired with this device we compared images of human capillary networks acquired with svOCT and fluorescein angiography. We found that the density of capillary microvasculature acquired with this svOCT device was visibly greater than fluorescein angiography. We also found that this svOCT device had the capacity to generate en face images of distinct capillary networks that are morphologically comparable with previously published histological studies. Finally, we found that this svOCT device has the ability to non-invasively illustrate the common manifestations of diabetic retinopathy and retinal vascular occlusion. The results of this study suggest that graphics processing unit accelerated svOCT has the potential to non-invasively provide useful quantitative information about human retinal capillary networks. Therefore svOCT may have clinical and research applications for the management of retinal microvascular diseases, which are a major cause of visual morbidity worldwide.

How Does Lowering of Intraocular Pressure Protect the Optic Nerve

Until recently, the evidence that lowering intraocular pressure (IOP) protects the optic nerve from glaucomatous damage was weak. Several randomized controlled trials have provided stronger evidence that lowering IOP prevents glaucomatous progression. Optic nerves appear to be highly variable in their susceptibility to raised IOP. Elevated IOP likely triggers several parallel, but interacting mechanisms, including direct axonal damage, failure of load-bearing tissues, and disturbances in microvascular supply. The cellular mechanisms that translate these mechanical and physiologic stresses and that lead to excavation of optic nerve tissue are beginning to be understood.