Blood Flow

Neurovascular Coupling, Hemodynamic Imaging, Imaging Pericytes

Objective measures of blood flow in the retina

The neurons of the retina are one of the most metabolically active tissues in the human body. To serve this demand, a network of capillaries delivers nutrients and removes waste products from the highly metabolic neurons. Previously, the fine details of this capillary network have been obscured by insufficient spatial and temporal resolution. In this line of work, we are developing AOSLO technology to objectively report capillary blood flow by imaging the movement of single blood cells as they flow through the capillary network. This direct and non-invasive assessment of retinal blood flow provides structural and functional information on the vascular network of the retina. We are pursuing several projects in this area:

  1. examining the role of capillary level neurovascular coupling in the central nervous system
  2. characterizing flow and structure parameters that describe normal vascular perfusion and
  3. exploring whether aberrant blood flow patterns can serve as a preclinical biomarker of early stages of retinal disease.


The primate microvascular network imaged without contrast agents. The movement of single blood cells as they traverse through the retinal circulation creates a pattern of motion in image sequences captured with an adaptive optics scanning laser ophthalmoscope (AOSLO). The light scatter and absorbance of moving blood cells creates a spatio-temporal flicker in images. The image sequences are evaluated for high spatio-temporal pixel flicker, which indicates particle movement (blood flow).

Non invasive imaging of the microvascular network

We are developing a new methodology which precludes the need for extrinsic contrast agents. Using the intrinsic spectral and scatter properties of individual white and red blood cells, we capture the movement of these cells in dynamic movies. The movement of these cells in an orderly pattern through the retinal circulation provides a motion contrast signal. This local motion is extracted from global motion in order to provide an intensity map of blood motion. This map reveals features of the retina with high motion (blood vessels) and low motion (static structures such as neurons of the retina). Therefore, the resultant perfusion maps not only provide structural information regarding the integrity of the microvasculature, but also functional information as it reports the dynamic nature of functional perfusion.


Blood flow in a single capillary path is imaged without contrast agents. Top A single blood cell travels through a capillary (dashed green trace). Movement of this cell over time is highlighted with yellow arrows. The centerline profile of the capillary is "flattened" to show displacement and plotted over time (spatio-temporal plot, bottom). Change in displacement, dx, divided by change in time, dt reveals the velocity of cells. Blood velocities ~0.3-3 mm/second were measured by this method. Image field is 60 µm wide in top images.

Objective measurements of blood flow

To date, few techniques are available to provide information on the flow velocity of blood in the retina. Conventional techniques such as laser Doppler flowmetry are limited in approach because they can only report velocity in the largest vessels of the eye. Moreover, they use an inferred method of reporting blood flow rather than a direct and objective measure of blood speed. In this project, we use a scanning system to directly measure the speed of individual blood cells as they move through the retinal circulation.


With AOSLO, we can observe three distinct capillary stratifications in the mouse retinal circulation. The above image shows a through-focus capture of three distinct capillary stratifications. Image field is 5 degrees of visual angle, ~150 microns across.

Confocal imaging in vivo

The AOSLO is a confocal instrument. By dynamically changing the depth of focus, we can optically section the retina. In the above image we can distinguish the location of three levels of capillaries in the retinal circulation.


Imaging at a deep focus, confocal AOSLO reveals the bore of the central retinal artery en face using NIR, 796nm light (bottom left). The diameter of the central retinal artery was measured with and without visual stimulation. Diameters were normalized to mean dark-adapted diameter. After presentation of flickering 5 hz, 514 nm light, there was a significant increase of central retinal artery diameter of over 3% (green trace). There was no dilation in the absence of visual stimulation (red trace).

Imaging blood flow dynamics in response to neural stimulation

In the central nervous system, increased levels of neural activity are associated with an increase in local blood flow, a response known as functional hyperemia. The circuit that mediates this local increase in blood flow is poorly understood because the neurons, glia and vascular cells reside deep in the opaque recesses of the brain. In this study, we attempt to image the neurovascular unit non-invasively in the retina, through the optical window of the eye.


Image shows extrinsically labeled pericytes for the Neural-glial antigen 2. Images are from A) confocal Scanning laser ophthalmoscope. B) 2-channel adaptive optics showing capillary perfusion (magenta) and NG2+ pericytes (green). C) High resolution image of a single AOSLO field. D) Validation of pericyte labeling in post-mortem histology.

Characterizing pericyte density and regulation of blood flow in the mouse retina in vivo

Pericytes are vascular associated cells that have been implicated as mediators of blood brain barrier formation, capillary structural scaffolds, and more recently, have implicated in regulating blood flow at the level of capillaries. However, many of these functions have been inferred from post mortem histology as single pericytes are very small (<7 microns in diameter) and offer poor optical contrast in the native retina. In this line of work, we are investigating the structural and functional roles of pericytes in the living animal. To overcome previous limitations of resolution and optical contrast we image NG2 transgenic mice which have fluorescent pericytes in the brain and retina with AOSLO technology. To image these fluorescent cells, we have equipped the AOSLO with a second channel that allows fluorescence imaging simultaneously with the infrared reflectance imaging described above. In this line of work, we are characterizing the role of pericytes in providing more blood to active neural regions (neurovascular coupling) and characterizing the role of pericyte loss in animal models of human diabetes.

Publications

Center for Visual Science University of Rochester