Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Summary

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Abstract

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier¹. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells². Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.

The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption³. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug⁴. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine⁵ and bradykinin⁶. This method, however, is selective primarily for the brain-tumor barrier⁷. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol⁸. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier⁹. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research^10-12.

In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N₂O anesthesia are used for their ability to maximize the effectiveness of barrier disruption¹³. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain ^8,13,14.

Protocol

1. Prepare Animal and Equipment for Procedure

1. Before beginning surgery, prepare the surgical area and the animal. Make the carotid catheter by inserting a 23-gauge blunt needle into one end of 12" of PE50 tubing. Cut an approximately 45° bevel in the opposite end of the catheter. Sterilize equipment prior to the procedure. Wear a hair bonnet, surgical mask, and sterile gloves.
2. Place a heating pad on the surface where the surgery will be performed. Activate it and allow it to warm. Cover the area with an absorbent bench pad.
3. Verify that you have 25% mannitol in solution without crystals. If crystals are present, dissolve them by ....

Results

Figure 1. Visualizing blood-brain barrier disruption via Evans blue dye extravasation. Evans blue dye is a pigment that binds to albumin and is not extravasated into the brain under physiological conditions. Disruption of the blood-brain barrier on one side of the br.......

Discussion

There are a few means of maximizing the efficacy of BBBD. It is important to minimize bleeding during the cut-down phase. Blood pressure and heart rate can be affected by substantial bleeding and these factors are known to affect the degree of BBBD¹³. Bleeding can be reduced by using sutures to ligate major vessels, such as the superior thyroid and occipital arteries, which must be divided. Additionally, electrocautery can be used to divide vessels and dissect areas that have a rich blood supply. It is also im.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Long Evans rat	Harlan Laboratories		210-250 g, male
PE 50 Tubing	Beckton-Dickinson
18 gauge x 2.5" IV catheter	Terumo		For ET tube
30" IV extension sets	Abbott
26 gauge veterinary IV catheter	Monoject
Evans blue dye	Sigma	E2129
Bipolar	Codman
Filter, 5 μm	Braun