Dynamic in vivo imaging of cerebral blood flow and blood-brain barrier permeability

Abstract

The brain is characterized by an extremely rich blood supply, regulated by changes in blood vessel diameter and blood flow, depending on metabolic demands. The blood-brain barrier (BBB)-a functional and structural barrier separating the intravascular and neuropil compartments-characterizes the brain's vascular bed and is essential for normal brain functions. Disruptions to the regional cerebral blood supply, to blood drainage and to BBB properties have been described in most common neurological disorders, but there is a lack of quantitative methods for assessing blood flow dynamics and BBB permeability in small blood vessels under both physiological and pathological conditions. Here, we present a quantitative image analysis approach that allows the characterization of relative changes in the regional cerebral blood flow (rCBF) and BBB properties in small surface cortical vessels. In experiments conducted using the open window technique in rats, a fluorescent tracer was injected into the tail vein, and images of the small vessels at the surface of the cortex were taken using a fast CCD camera. Pixel-based image analysis included registration and characterization of the changes in fluorescent intensity, followed by cluster analysis. This analysis enabled the characterization of rCBF in small arterioles and venules and changes in BBB permeability. The method was implemented successfully under experimental conditions, including increased rCBF induced by neural stimulation, bile salt-induced BBB breakdown, and photothrombosis-mediated local ischemia. The new approach may be used to study changes in rCBF, neurovascular coupling and BBB permeability under normal and pathological brain conditions.

Figures

Fig. 1

Flow visualization in pial vessels and image analysis: (A–C) Intravenous injection of the fluorescent tracer LY under control conditions resulted in an early increase in signal intensity within the surface arterioles (A), followed by delayed labeling of the venules (B). By using image analysis, an intensity–time curve was created (C), reflecting the change in tracer intensity within the local vasculature. The curve allows the estimation of several parameters using a segmented linear model (inset—red line, blue line—the raw data in a representative pixel). (D) Analysis results are represented for each voxel of the original image, enabling the distinction between defined vessels and the extravascular brain tissue. (E) Cluster analysis was used for automatic clustering of similarly behaving pixels (see Methods). Left image is the result of setting the number of clusters to 2; pixels are labeled as red for presumed blood vessels and gray for extravascular tissue. Right image—number of clusters was set to 3, allowing the distinction between arterioles (red), venules (blue) and tissue (gray). Light gray—the original signal in the extravascular compartment, dark gray—corrected signal following “background reduction” (see text).

Fig. 2

Experimental CBF increases following stimulation of the ethmoidal nerve: (A) A fluorescent tracer (LY) was injected under control conditions (left image) and at the end of each stimulus train (right image at 3 mA). Vasodilatation of surface vessels can clearly be seen. (B) LDF showed increased rCBF in each stimulation train above 1mA (inset). The graph shows mean measurements of maximal rCBF increase during each stimulation train, showing a consistent increase in mean rCBF when stimulation was repeated at 2 mA but not at 1mA (n = 5 at each intensity). (C) Stimulation of the ethmoidal nerve induced increase in rCBF, which was associated with intensity-dependent dilatation of arterioles. (D) Results of dynamic analysis during tracer injection are shown for max value, demonstrating an increasing change in maximal intensity (i.e., flow) following stimulation. (E) Mean intensity changes during injection in the arterial (left) and venous (right) compartments. Note that a significant reduction in tti and increases in incline and max values were observed at stimulation intensities higher than 1 mA.

Fig. 3

Imaging analysis reveals increased BBB permeability: (A–B) Fluorescent images of surface vessels during the venous phase of injection before (A) and after (B) DOC treatment. Diffusion of the injected tracer (LY) outside the vessels is seen after treatment, indicating BBB breakdown. (C) A coronal section following treatment with DOC and injection of Evans blue demonstrates extravasation of the albumin-binding dye into the treated cortical tissue, which indicates BBB breakdown. (D) Treatment with DOC was associated with a steady increase in LDF signal (left) and vasodilatation. The bar graph shows the number of pixels in the arterial and venous clusters under control conditions (ACSF) and following perfusion with DOC. (E) Maps of mean transient time (MTT) showing decreased MTT in arterioles and the robust increase in the extravascular space where the tracer was accumulating. (F) Intensity curve for each compartment showing the increased flow in the arterial compartment together with decreased signal intensity in the venous compartment after DOC (dashed line). Note the robust slowing of the signal decay in the extravascular compartment after BBB breakdown.

Fig. 4

Intravascular thrombosis and experimental ischemia: (A, B) Fluorescent images of the arterial (left) and venous (right) phases before (A: control) and 30 min after (B) RBG injection and cortical exposure to light. The thrombotic vessels is observed as an abrupt stop of blood supply (“center”). Note the intact blood supply in the surrounding brain tissue (“surround”). (C) MTT maps before and after the induction of photo-thrombosis, showing the robust reduction in MTT in the ischemic region and the increased MTT in the surrounding (“penumbra”4) region. (D) Intensity curve for each compartment, showing the reduction in the perfusion in the ischemic core. In the surrounding brain a clear increase in signal is noted in the arterial, venous and extravascular compartments, suggesting increased blood flow and BBB breakdown. Inset: left: brain tissue 1 h after RBG treatment (30 min after injection of Evans blue)—confirmed BBB breakdown in the region surrounding the thrombotic vessel. Right: histological brain section (cresyl violet staining) 21 days after a photothrombotic lesion.