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.
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 research10-12.
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.
1. Prepare Animal and Equipment for Procedure
2. Intubate the Rat
3. Establish Propofol IV Anesthesia
4. Expose the Bifurcation of the Common Carotid Artery
5. Catheterize the External Carotid Artery
6. Administer Mannitol
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 brain allows Evans blue to enter and stain the disrupted hemisphere blue while the non-disrupted hemisphere remains unchanged. Thus, it is a useful marker of blood-brain barrier disruption. Figure 1A is an example of a brain without blood-brain barrier disruption. Figure 1B is an example of a brain stained blue after osmotic BBBD using 25% mannitol. Note that one hemisphere is stained blue while the other remains unstained. Some blue has entered the left hemisphere near the medial longitudinal fissure as a result of mixing of blood flow at the Circle of Willis.
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 important to keep air and solids out of all lines placed into the animal – particularly the carotid catheter. Working quickly with the mannitol helps to ensure that crystals do not form inside the syringe or tubing.
It is important to use a mannitol flow rate that is effective for the size and strain of rat used in this experiment. The flow rate of the mannitol infusion must match the pressure in the rat’s common carotid artery such that a high concentration of mannitol flows into the ICA without flowing down the common carotid into the heart and lungs, which can cause damage. Researchers choosing a different size and strain of rat than the one used in this study should conduct experiments to find the correct mannitol flow rate.
One of the limitations of this procedure is that it requires the sacrifice of the ECA. Once the ECA is catheterized, it becomes difficult and unreliable to catheterize again. Thus, this procedure cannot be performed twice on the same hemisphere. Additionally, compounds given following BBBD must be carefully tested for neurotoxicity. Some drugs that are ordinarily safe become dangerous once they cross the blood-brain barrier.
Despite these limitations, BBBD has been used extensively to study improved delivery of chemotherapy to brain tumors8,15-18. It may also be useful for other therapeutic applications in which a drug does not effectively cross the blood-brain barrier and administering mannitol is known to be safe.
The authors have nothing to disclose.
This work was supported by the J.B. Marshall Foundation.
Material Name | Company | Catalogue number | Comment |
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 |