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Far-red tracer analysis of traumatic cerebrovascular permeability

Significance statement

 

The microvasculature in the brain is unique due to the presence of the blood brain barrier (BBB). Increased BBB permeability and cerebral edema are important therapeutic targets in traumatic brain injury research pertaining to the secondary phase of injury. Current assays evaluating permeability and edema, such as Evans Blue, are limited as extraparenchymal and intravascular dye cannot be easily excluded from intraparenchymal dye due to the extraction process where tissue structure is also lost. Our study evaluated Alexa Fluor 680, a far-red dye bioconjugated to 10kDa dextran as an in vivo alternative to albumin (66 kDa) bound Evans Blue in a rat model of traumatic brain injury. We found that Alexa Fluor 680 allowed us to move towards a multimodal assay strategy where (1) the sensitivity to BBB perturbations was increased due to smaller marker size, (2) penumbral at-risk areas to microvascular injury were visually identified and quantified, and (3) tissue architecture was preserved for additional imaging studies. We believe that this assay platform improves brain injury research and can also be easily applied to other preclinical models and organs systems where precise regional changes in tissue microvasculature permeability and edema are important for diagnosis, intervention and outcome.

Figure legend: A representative coronal section from a rat brain 72 hours status post controlled cortical impact injury.  Cerebral edema can be visualized by the localized Alexa Fluor 680 dye, shown in red, in the injured brain parenchyma after passing through the blood brain barrier of the neurovasculature (left). Threshold analysis can be used to identify penumbral at-risk areas, marked in red, where early blood brain barrier compromise may be occurring (right)

Far-red tracer analysis of traumatic cerebrovascular permeability

 

 

 

 

 

Journal Reference

Liao GP1, Olson SD2, Kota DJ2, Hetz RA2, Smith P2, Bedi S2, Cox CS Jr2. J Surg Res. 2014 Aug;190(2):628-33.

1Department of Pediatric Surgery, University of Texas Health Science Center at Houston, Houston, Texas. Electronic address: [email protected] and

2Department of Pediatric Surgery, University of Texas Health Science Center at Houston, Houston, Texas.

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 aftertraumatic brain injury and maybe particularly useful in demonstrating BBB preservation of at-risk regions in response to therapeutic agents.

Copyright © 2014 Elsevier Inc. All rights reserved.

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