Far-red tracer analysis of traumatic cerebrovascular permeability

Abstract

Background: Blood brain barrier (BBB) compromise is a key pathophysiological component of secondary traumatic brain injury characterized by edema and neuroinflammation in a previously immune-privileged environment. Current assays for BBB permeability are limited by working size, harsh extraction processes, suboptimal detection via absorbance, and wide excitation fluorescence spectra. In this study, we evaluate the feasibility of Alexa Fluor 680, a far-red dye bioconjugated to dextran, as an alternative assay to improve resolution and sensitivity.

Methods: Alexa Fluor was introduced intravenously on the day of sacrifice to three groups: sham, controlled cortical impact (CCI), and CCI treated with a cell based therapy known to reduce BBB permeability. The brains were sectioned coronally and imaged using an infrared laser scanner to generate intensity plot profiles as well as signal threshold images to distinguish regions with varying degrees of permeability.

Results: Linear plot profile analysis demonstrated greater signal intensity from CCI than treated rats at corresponding injury depths. Threshold analysis identified rims of signal at low + narrow threshold ranges. The integrated signals from a treatment group known to preserve the BBB were significantly less than the groups with CCI injury alone. There was no significant difference at high + wide signal intensity threshold ranges.

Conclusions: Alexa Fluor 680 infrared photodetection and image analysis can aid in detecting differential degrees of BBB permeability after traumatic brain injury and maybe particularly useful in demonstrating BBB preservation of at-risk regions in response to therapeutic agents.

Keywords: Blood brain barrier; Edema; Fluorescence; Permeability; Traumatic brain injury.

Figure 1

Non-specific signals are reduced with Alexa Fluor compared to Evans Blue dye as evidenced by the high signal from sham animals receiving Evans Blue. Evans Blue CCI (n=11), Evans Blue Sham (n=6), Alexa Fluor CCI (n=9), Alexa Fluor Sham (n=10). The Y axis OD/Wt refers to optical density (absorbance) per rat weight. The X axis Integrated Density refers to the cumulated signal detected by the far-red scanner across the brain slices.

Figure 2

A representative montage containing the three experimental groups after merging the two detection channels (700nm - Alexa 680 and 800nm - background). Red areas represent presence of Alexa-Fluor 680 dye.

Figure 3

One dimensional signal intensity along the impact trajectory represented as an orthogonal line connecting the cortical surface to the 3^rd ventricle. The signal intensity can serve as a surrogate for blood brain barrier permeability along the depth of injury.

Figure 4

The same representative slice from each of the three experimental groups used in Figure 1 with the application of 4 different intensity thresholds to represent the three low + narrow threshold ranges corresponding to penumbral areas and the one high + wide threshold range representing foci of hemorrhage/contusion/complete BBB disruption. The integrated signals from the treated rats were significantly less than CCI rats, suggesting a therapeutic effect on the penumbral regions.

Figure 5

A scatter plot representation of Figure 4 demonstrates integrated signal across each threshold intensity range for the three experimental groups. Despite the wide spread within each treatment group suggesting heterogeneity of injury, there is a consistent trend within each treatment group from the three low + narrow threshold ranges to the one high + wide threshold range. A significant therapeutic effect is seen between treated versus CCI rats in the low + narrow threshold ranges, which correspond to the red rims identified in Figure 4 (likely at-risk penumbral regions of BBB permeability).