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نمایش مقاله

Title:

Evaluation of Ultrasound-Mediated Blood-Brain Barrier Disruption Using Evans Blue in Rats

Subject:

Clinical Applications of Brain Mappin

Presenter Author:
Name:	Ahlam Almanie

Author:
Name 1:	Ahlam Almanie - Presenter
Affiliation 1:	Department of Medical Physics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

Name 2:	Manijhe Mokhtari Dizaji - Corresponding
Affiliation 2:	Department of Medical Physics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

Name 3:	Zeinab Shankayi
Affiliation 3:	Department of Medical Physics, Faculty of Medical Sciences, Baqiyatallah University of Medical Sciences, Tehran, Iran

Name 4:	Zeinab Hormozi-Moghaddam
Affiliation 4:	Department of Radiology, Faculty of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran

Name 5:	Fatemeh Ghaderi
Affiliation 5:	Doctor of Veterinary Medicine, Faculty of Veterinary Medicine, Semnan University, Semnan, Iran

Name 6:	Razieh Zaghian
Affiliation 6:	Department of Radiotherapy Oncology, Shohadaye Tajrish Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran

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Abstract

Original Research

introduction :

The blood-brain barrier (BBB) is a selective physiological barrier that restricts the passage of most macromolecules and micromolecular drugs, including chemotherapy agents, from reaching the brain. Low-intensity ultrasound combined with microbubbles (MB) has emerged as a promising, non-invasive method to transiently disrupt the BBB and facilitate drug delivery. In this study, we compared the effects of different ultrasound irradiation frequencies and intensities on BBB permeability using Evans Blue (EB) dye as a marker. EB is a dye that normally does not cross the intact BBB; its presence in brain tissue indicates increased permeability.

Methods:

Adult wistar rats allocated into four groups: 1) a control group receiving only EB without ultrasound, 2) MB injection with 1 MHz ultrasound at 2 W/cm², 3) MB injection with 40 kHz ultrasound at 0.34 W/cm², and 4) a dual-frequency group receiving both 1 MHz and 40 kHz ultrasound as in groups 2 and 3. After anesthesia induction using ketamine and xylazine, microbubbles were injected intravenously, followed immediately by ultrasound application except in controls. Based on prior studies, maximum BBB opening occurs about 30 minutes after ultrasound. Thus, EB dye was administered 30 minutes post-irradiation. Visible blue staining of the rats’ eyes, nose, and skin confirmed systemic distribution of EB. Brains were collected 30 minutes after EB injection, homogenized, treated with trichloroacetic acid to precipitate proteins, and centrifuged to obtain supernatant for spectrophotometric analysis. EB extravasation was quantified by optical density at 620 nm.

Results:

Result showed that dual-frequency ultrasound significantly enhanced BBB permeability, increasing EB penetration by 42% over controls, 17% more than the 40 kHz group, and 35% more than the 1 MHz group. The 1 MHz single frequency showed the smallest increase in permeability, likely related to its lower mechanical index (MI=0.25) compared to the higher MI for 40 kHz (MI=0.55). Statistical analysis confirmed all ultrasound groups had significantly greater BBB permeability than controls.

Conclusion:

These findings suggest that the combination of dual-frequency ultrasound irradiation provides superior BBB opening and may optimize delivery of therapeutic agents to the brain. This approach holds potential for improving treatment strategies for central nervous system disorders where drug delivery is currently limited by the BBB.

Keywords:

Blood-Brain Barrier, Ultrasound, Microbubble, Evans Blue