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High and Low Molecular Weight Fluorescein Isothiocyanate (FITC)-Dextrans to Assess Blood-Brain Barrier Disruption: Technical Considerations.

Hoffmann A, Bredno J, Wendland M, Derugin N, Ohara P, Wintermark M - Transl Stroke Res (2010)

Bottom Line: By using appropriate preparation methods, false negative results can be minimized.If exposed directly to saline, the signal intensity of these FITC-dextrans decreased.Diffusion and washout of low molecular weight FITC-dextran can be avoided by direct immobilization through immediate freezing of the tissue.

View Article: PubMed Central - PubMed

ABSTRACT
This note is to report how histological preparation techniques influence the extravasation pattern of the different molecular sizes of fluorescein isothiocyanate (FITC)-dextrans, typically used as markers for blood-brain barrier leakage. By using appropriate preparation methods, false negative results can be minimized. Wistar rats underwent a 2-h middle cerebral artery occlusion and magnetic resonance imaging. After the last imaging scan, Evans blue and FITC-dextrans of 4, 40, and 70 kDa molecular weight were injected. Different histological preparation methods were used. Sites of blood-brain barrier leakage were analyzed by fluorescence microscopy. Extravasation of Evans blue and high molecular FITC-dextrans (40 and 70 kDa) in the infarcted region could be detected with all preparation methods used. If exposed directly to saline, the signal intensity of these FITC-dextrans decreased. Extravasation of the 4-kDa low molecular weight FITC-dextran could only be detected using freshly frozen tissue sections. Preparations involving paraformaldehyde and sucrose resulted in the 4-kDa FITC-dextran dissolving in these reactants and being washed out, giving the false negative result of no extravasation. FITC-dextrans represent a valuable tool to characterize altered blood-brain barrier permeability in animal models. Diffusion and washout of low molecular weight FITC-dextran can be avoided by direct immobilization through immediate freezing of the tissue. This pitfall needs to be known to avoid the false impression that there was no extravasation of low molecular weight FITC-dextrans.

No MeSH data available.


Related in: MedlinePlus

Representing images showing the leakage pattern of Evans blue-tagged albumin and of the 4-kDa FITC–dextran in the infarction core after different histologic processing procedures on the sides ipsilateral and contralateral to the infarct. On the ischemic side, the extravasation of Evans blue-tagged albumin resulted in diffuse and a punctuated distribution (cellular uptake) of the red fluorescence. a, e If the brain was frozen directly, free Evans blue-tagged albumin was more important. i On the nonischemic side, Evans blue-tagged albumin remains in the vessel lumen. c, g, l The appearance of the 4-kDa FITC–dextran for the same fields depends upon the processing method. On the ipsilateral side, using preparation #2 (PFA + sucrose) and #3 (PFA + freezing), no leakage of 4 kDa FITC–dextran can be detected. b, f If frozen directly, a bright green fluorescent cloud diffuses out at the site of the BBB opening. k On the contralateral side, 4 kDa FITC–dextran can be detected intravascularly when PFA + sucrose (preparation #1) or only PFA are used (preparation #2). d, h A 20-μm space bar appears in m and holds for all the images
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Fig3: Representing images showing the leakage pattern of Evans blue-tagged albumin and of the 4-kDa FITC–dextran in the infarction core after different histologic processing procedures on the sides ipsilateral and contralateral to the infarct. On the ischemic side, the extravasation of Evans blue-tagged albumin resulted in diffuse and a punctuated distribution (cellular uptake) of the red fluorescence. a, e If the brain was frozen directly, free Evans blue-tagged albumin was more important. i On the nonischemic side, Evans blue-tagged albumin remains in the vessel lumen. c, g, l The appearance of the 4-kDa FITC–dextran for the same fields depends upon the processing method. On the ipsilateral side, using preparation #2 (PFA + sucrose) and #3 (PFA + freezing), no leakage of 4 kDa FITC–dextran can be detected. b, f If frozen directly, a bright green fluorescent cloud diffuses out at the site of the BBB opening. k On the contralateral side, 4 kDa FITC–dextran can be detected intravascularly when PFA + sucrose (preparation #1) or only PFA are used (preparation #2). d, h A 20-μm space bar appears in m and holds for all the images

Mentions: Animals, which had been injected with the 4-kDa dextran did not show any apparent leakage of FITC–dextran if fixed by immersion in paraformaldehyde and sucrose (preparations #1 and #2) or just in paraformaldehyde (preparation #3). Interestingly, for all preparations, 4 kDa dextran could be identified within the lumen of the vessels on the contralateral side to the ischemic lesions. When freshly frozen sections were used (preparation #4), leakage of the 4-kDa dextran could be visualized on the infarcted ipsilateral side. Compared to the larger molecular weight dextrans, the extravasated 4-kDa dextran tends to spread out further away from the vessels into the extravascular space (Table 2; Fig. 3).Table 2


High and Low Molecular Weight Fluorescein Isothiocyanate (FITC)-Dextrans to Assess Blood-Brain Barrier Disruption: Technical Considerations.

Hoffmann A, Bredno J, Wendland M, Derugin N, Ohara P, Wintermark M - Transl Stroke Res (2010)

Representing images showing the leakage pattern of Evans blue-tagged albumin and of the 4-kDa FITC–dextran in the infarction core after different histologic processing procedures on the sides ipsilateral and contralateral to the infarct. On the ischemic side, the extravasation of Evans blue-tagged albumin resulted in diffuse and a punctuated distribution (cellular uptake) of the red fluorescence. a, e If the brain was frozen directly, free Evans blue-tagged albumin was more important. i On the nonischemic side, Evans blue-tagged albumin remains in the vessel lumen. c, g, l The appearance of the 4-kDa FITC–dextran for the same fields depends upon the processing method. On the ipsilateral side, using preparation #2 (PFA + sucrose) and #3 (PFA + freezing), no leakage of 4 kDa FITC–dextran can be detected. b, f If frozen directly, a bright green fluorescent cloud diffuses out at the site of the BBB opening. k On the contralateral side, 4 kDa FITC–dextran can be detected intravascularly when PFA + sucrose (preparation #1) or only PFA are used (preparation #2). d, h A 20-μm space bar appears in m and holds for all the images
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037479&req=5

Fig3: Representing images showing the leakage pattern of Evans blue-tagged albumin and of the 4-kDa FITC–dextran in the infarction core after different histologic processing procedures on the sides ipsilateral and contralateral to the infarct. On the ischemic side, the extravasation of Evans blue-tagged albumin resulted in diffuse and a punctuated distribution (cellular uptake) of the red fluorescence. a, e If the brain was frozen directly, free Evans blue-tagged albumin was more important. i On the nonischemic side, Evans blue-tagged albumin remains in the vessel lumen. c, g, l The appearance of the 4-kDa FITC–dextran for the same fields depends upon the processing method. On the ipsilateral side, using preparation #2 (PFA + sucrose) and #3 (PFA + freezing), no leakage of 4 kDa FITC–dextran can be detected. b, f If frozen directly, a bright green fluorescent cloud diffuses out at the site of the BBB opening. k On the contralateral side, 4 kDa FITC–dextran can be detected intravascularly when PFA + sucrose (preparation #1) or only PFA are used (preparation #2). d, h A 20-μm space bar appears in m and holds for all the images
Mentions: Animals, which had been injected with the 4-kDa dextran did not show any apparent leakage of FITC–dextran if fixed by immersion in paraformaldehyde and sucrose (preparations #1 and #2) or just in paraformaldehyde (preparation #3). Interestingly, for all preparations, 4 kDa dextran could be identified within the lumen of the vessels on the contralateral side to the ischemic lesions. When freshly frozen sections were used (preparation #4), leakage of the 4-kDa dextran could be visualized on the infarcted ipsilateral side. Compared to the larger molecular weight dextrans, the extravasated 4-kDa dextran tends to spread out further away from the vessels into the extravascular space (Table 2; Fig. 3).Table 2

Bottom Line: By using appropriate preparation methods, false negative results can be minimized.If exposed directly to saline, the signal intensity of these FITC-dextrans decreased.Diffusion and washout of low molecular weight FITC-dextran can be avoided by direct immobilization through immediate freezing of the tissue.

View Article: PubMed Central - PubMed

ABSTRACT
This note is to report how histological preparation techniques influence the extravasation pattern of the different molecular sizes of fluorescein isothiocyanate (FITC)-dextrans, typically used as markers for blood-brain barrier leakage. By using appropriate preparation methods, false negative results can be minimized. Wistar rats underwent a 2-h middle cerebral artery occlusion and magnetic resonance imaging. After the last imaging scan, Evans blue and FITC-dextrans of 4, 40, and 70 kDa molecular weight were injected. Different histological preparation methods were used. Sites of blood-brain barrier leakage were analyzed by fluorescence microscopy. Extravasation of Evans blue and high molecular FITC-dextrans (40 and 70 kDa) in the infarcted region could be detected with all preparation methods used. If exposed directly to saline, the signal intensity of these FITC-dextrans decreased. Extravasation of the 4-kDa low molecular weight FITC-dextran could only be detected using freshly frozen tissue sections. Preparations involving paraformaldehyde and sucrose resulted in the 4-kDa FITC-dextran dissolving in these reactants and being washed out, giving the false negative result of no extravasation. FITC-dextrans represent a valuable tool to characterize altered blood-brain barrier permeability in animal models. Diffusion and washout of low molecular weight FITC-dextran can be avoided by direct immobilization through immediate freezing of the tissue. This pitfall needs to be known to avoid the false impression that there was no extravasation of low molecular weight FITC-dextrans.

No MeSH data available.


Related in: MedlinePlus