Name: Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics (Video) | JoVE Journal
Uploaded: 2012-11-30T12:00:00.000+00:00
Duration: 8 min 43 s
Description: Learn to disrupt the blood-brain barrier in rats using intra-arterial mannitol infusion via external carotid catheterization. This method enables targeted neurovascular drug delivery confirmed by Evan's Blue extravasation.

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5 minutes

This work was supported by the J.B. Marshall Foundation.

1. Prepare Animal and Equipment for Procedure
<ol>
	<li>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&deg; bevel in the opposite end of the catheter. Sterilize equipment prior to the procedure. Wear a hair bonnet, surgical mask, and sterile gloves.</li>
	<li>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.</li>
	<li>Verify that you have 25% mannitol in solution without crystals. If crystals are present, dissolve them by placing the vial in a hot water bath at 80 &deg;C and agitating periodically.</li>
	<li>Record the mass of the animal.</li>
	<li>Make a propofol dilution for delivery at 0.8 mg/kg/min at a rate of 0.1 ml/min.</li>
	<li>Place the rat in the induction box for 3 min. Settings: 5% isoflurane, 2 L/min flow rate on ventilator.</li>
	<li>On top of a disposable towel, shave the neck and both inner thighs.</li>
</ol>
2. Intubate the Rat
<ol>
	<li>Place the animal back in the induction box for 3 min.</li>
	<li>Prepare your endotracheal tube with an IV catheter. For this size and strain of rat, we use an 18 ga catheter. The tube must be large enough to allow adequate airflow, but small enough to fit into the trachea without causing damage. Pull the needle out of the plastic catheter and carefully break the needle approx. 1 cm from its sharp tip. The bunted needle will be your stylus. Put the needle back into the catheter.</li>
	<li>Hang the rat by its teeth on an angled surface.</li>
	<li>Shine a light directly against the animal's neck and lined up between its ears.</li>
	<li>Insert a small spatula approx. 3 cm into the oropharynx. Lift upward while gently pulling the tongue out of the mouth.</li>
	<li>Visualize the opening of the trachea. You will see a small hole open and close with each breath.</li>
	<li>Gently insert catheter into the hole. You can sometimes feel the plastic enter the cartilage of the trachea. The trachea is sensitive. Do not force the catheter.</li>
	<li>Set the ventilator to 1 L/min and 60 bpm and the isoflurane to 2%.</li>
	<li>Connect the catheter to the ventilator and tape the tube to the operating surface.</li>
	<li>Verify the animal is anesthetized by performing a toe pinch.</li>
</ol>
3. Establish Propofol IV Anesthesia
<ol>
	<li>Secure the rat by taping all four limbs and the tail to the operating surface.</li>
	<li>Scrub the shaved neck and groin sites with chlorhexidine soap three times. Then scrub with chlorhexidine rinse three times.</li>
	<li>Attach a syringe of propofol to an IV extension set. Mount the syringe on an infusion pump.</li>
	<li>Using a scalpel, cut through the skin of the shaved thigh. Avoid cutting muscle.</li>
	<li>Open the incision with forceps to visualize the femoral vein.</li>
	<li>Pull away tissue over the artery to expose the vessel.</li>
	<li>Insert a 26 ga monoject veterinary IV catheter into vein. When the needle punctures the wall of the vein you will feel it become easier to push. Retract the needle and push the catheter into the vessel.</li>
	<li>Start the propofol infusion at 0.1 ml/min.</li>
	<li>3-5 min after beginning the propofol infusion, switch the anesthesia gas to a mixture of 50% O2 and 50% N2O and shut off the isoflurane.</li>
</ol>
4. Expose the Bifurcation of the Common Carotid Artery
<ol>
	<li>Make a midline incision at the neck with scalpel blade.</li>
	<li>Cut between the two large salivary glands with scissors or scalpel.</li>
	<li>Dissect away tissue lateral to the midline to expose the carotid artery. Use forceps to open layers of tissue. You can use scissors to remove pieces that you expose with the forceps and determine to be minimally vascular. Control bleeding with gauze, cotton swabs, and cautery if necessary.</li>
	<li>Inject 3 ml saline IP if there has been moderate to heavy bleeding.</li>
	<li>Use retractors to pull tissue away from the carotid. Find the bifurcation.</li>
	<li>Expose the occipital artery just distal to the bifurcation. Cauterize it between forceps and separate it with an electrocautery.</li>
	<li>Use forceps to fully expose the carotid bifurcation and as much of the ECA as possible.</li>
	<li>Expose the superior thyroid artery branching off the ECA distal to the occipital artery. Cauterize it between forceps and separate it with an electrocautery.</li>
</ol>
5. Catheterize the External Carotid Artery
<ol>
	<li>Place 2 lengths of 4/0 silk under the ECA.</li>
	<li>Grab 1 length of suture with your hemostat and retract the ECA back caudally (toward the rat's feet).</li>
	<li>Take the other suture and tie as far cranially (toward the rat's mouth) as possible with a surgeon's knot. This forms a permanent ligature to sacrifice the ECA.</li>
	<li>Remove the first hemostat (the one that was applying caudal tension) and grab the other suture (the one that is knotted around the ECA) and apply gentle tension cranially with the hemostat.</li>
	<li>Tie another surgeon's knot loosely (do not secure - leave barely tied) distal to the bifurcation and proximal to the permanent ligature.</li>
	<li>Irrigate with 1% lidocaine to relax the artery.</li>
	<li>Fill your catheter with heparinized saline.</li>
	<li>Place a temporary vessel clip as close as possible to the bifurcation. Place vessel clip at an angle to maximize space for catheterization. Slide the loose knot toward the clip.</li>
	<li>Make a small arteriotomy (cut) in ECA just proximal to the ligature using microscissors. There should be no bleeding if the clip and ligature are secure.</li>
	<li>Using forceps to control the ECA walls, feed the beveled end of the catheter into the vessel.</li>
	<li>Secure the catheter with the loose suture knot. Tie the artery around the catheter.</li>
	<li>Remove the clip and clean the area with a cotton swab.</li>
	<li>Advance the catheter to about 1 mm distal to bifurcation. Hold the artery with forceps proximal to the knot to ensure the vessel is immobilized.</li>
</ol>
6. Administer Mannitol
<ol>
	<li>Fill an 8" IV extension set and a 6 ml syringe with mannitol without creating any air bubbles. Store this in the incubator at 37 &deg;C. Note: work quickly with mannitol - it will readily precipitate out of solution at room temperature.</li>
	<li>In order to visualize BBBD, you may administer Evans blue dye and watch it enter the areas of the brain where the barrier has been disrupted. To do this, give the animal 2 ml/kg of 2% Evans Blue through a new IV site on leg contralateral to the propofol infusion.</li>
	<li>Attach the extension set to a 5 &mu;m filter and fill it with mannitol to remove air bubbles. Then, attach this to the stopcock without creating bubbles.</li>
	<li>Administer mannitol at 0.09 ml/sec for 25 sec. Watch for the mannitol to reach equilibrium with the flow in common carotid at a point just proximal to the ICA. It should appear to pulse with the rat's heart rate. If the mannitol simply flows down toward the heart you have overcome the rat's blood pressure. This can cause life-threatening complications and poor BBBD.</li>
</ol>

<ol><li>Abbott, N. J., Patabendige, A. A. K., Dolman, D. E. M., Yusof, S. R., Begley, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+and+function+of+the+blood-brain+barrier%5BTitle%5D)+AND+%22Neurobiology+of+disease%22%5BJournal%5D)">Structure and function of the blood-brain barrier.</a> Neurobiology of disease. 37, 13-25 (2010).</li>
<li>Smith, Q. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Blood-Brain+Barrier%5BTitle%5D)+AND+%22%22%5BJournal%5D)">The Blood-Brain Barrier.</a> 89, 193-208 (2003).</li>
<li>Kroll, R. A., Neuwelt, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Outwitting+the+blood-brain+barrier+for+therapeutic+purposes%3A+osmotic+opening+and+other+means%5BTitle%5D)+AND+%22Neurosurgery%22%5BJournal%5D)">Outwitting the blood-brain barrier for therapeutic purposes: osmotic opening and other means.</a> Neurosurgery. 42, 1083(1998).</li>
<li>Kroll, R. A., Pagel, M. A., Muldoon, L. L., Roman-Goldstein, S., Neuwelt, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increasing+volume+of+distribution+to+the+brain+with+interstitial+infusion%3A+dose%2C+rather+than+convection%2C+might+be+the+most+important+factor%5BTitle%5D)+AND+%22Neurosurgery%22%5BJournal%5D)">Increasing volume of distribution to the brain with interstitial infusion: dose, rather than convection, might be the most important factor.</a> Neurosurgery. 38, 746-754 (1996).</li>
<li>Butt, A. M., Jones, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+histamine+and+antagonists+on+electrical+resistance+across+the+blood-brain+barrier+in+rat+brain-surface+microvessels%5BTitle%5D)+AND+%22Brain+research%22%5BJournal%5D)">Effect of histamine and antagonists on electrical resistance across the blood-brain barrier in rat brain-surface microvessels.</a> Brain research. 569, 100-105 (1992).</li>
<li>Inamura, T., Black, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bradykinin+selectively+opens+blood-tumor+barrier+in+experimental+brain+tumors%5BTitle%5D)+AND+%22Journal+of+Cerebral+Blood+Flow+%26+Metabolism%22%5BJournal%5D)">Bradykinin selectively opens blood-tumor barrier in experimental brain tumors.</a> Journal of Cerebral Blood Flow & Metabolism. 14, 862-870 (1994).</li>
<li>Matsukado, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+tumor+uptake+of+carboplatin+and+survival+in+glioma-bearing+rats+by+intracarotid+infusion+of+bradykinin+analog%2C+RMP-7%5BTitle%5D)+AND+%22Neurosurgery%22%5BJournal%5D)">Enhanced tumor uptake of carboplatin and survival in glioma-bearing rats by intracarotid infusion of bradykinin analog, RMP-7.</a> Neurosurgery. 39, 125(1996).</li>
<li>Kroll, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improving+drug+delivery+to+intracerebral+tumor+and+surrounding+brain+in+a+rodent+model%3A+a+comparison+of+osmotic+versus+bradykinin+modification+of+the+blood-brain+and%2For+blood-tumor+barriers%5BTitle%5D)+AND+%22Neurosurgery%22%5BJournal%5D)">Improving drug delivery to intracerebral tumor and surrounding brain in a rodent model: a comparison of osmotic versus bradykinin modification of the blood-brain and/or blood-tumor barriers.</a> Neurosurgery. 43, 879(1998).</li>
<li>Kinoshita, M., McDannold, N., Jolesz, F. A., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Noninvasive+localized+delivery+of+Herceptin+to+the+mouse+brain+by+MRI-guided+focused+ultrasound-induced+blood-brain+barrier+disruption%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Noninvasive localized delivery of Herceptin to the mouse brain by MRI-guided focused ultrasound-induced blood-brain barrier disruption.</a> Proceedings of the National Academy of Sciences. 103, 11719-11723 (2006).</li>
<li>Neuwelt, E. A., Dahlborg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemotherapy+administered+in+conjunction+with+osmotic+blood-brain+barrier+modification+in+patients+with+brain+metastases%5BTitle%5D)+AND+%22Journal+of+neuro-oncology%22%5BJournal%5D)">Chemotherapy administered in conjunction with osmotic blood-brain barrier modification in patients with brain metastases.</a> Journal of neuro-oncology. 4, 195-207 (1987).</li>
<li>Doolittle, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Safety+and+efficacy+of+a+multicenter+study+using+intraarterial+chemotherapy+in+conjunction+with+osmotic+opening+of+the+blood-brain+barrier+for+the+treatment+of+patients+with+malignant+brain+tumors%5BTitle%5D)+AND+%22Cancer%22%5BJournal%5D)">Safety and efficacy of a multicenter study using intraarterial chemotherapy in conjunction with osmotic opening of the blood-brain barrier for the treatment of patients with malignant brain tumors.</a> Cancer. 88, 637-647 (2000).</li>
<li>Guillaume, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intra-arterial+chemotherapy+with+osmotic+blood-brain+barrier+disruption+for+aggressive+oligodendroglial+tumors%3A+results+of+a+phase+I+study%5BTitle%5D)+AND+%22Neurosurgery%22%5BJournal%5D)">Intra-arterial chemotherapy with osmotic blood-brain barrier disruption for aggressive oligodendroglial tumors: results of a phase I study.</a> Neurosurgery. 66, 48(2010).</li>
<li>Remsen, L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+anesthetic+choice%2C+PaCO2%2C+and+other+factors+on+osmotic+blood-brain+barrier+disruption+in+rats+with+brain+tumor+xenografts%5BTitle%5D)+AND+%22Anesthesia+%26+Analgesia%22%5BJournal%5D)">The influence of anesthetic choice, PaCO2, and other factors on osmotic blood-brain barrier disruption in rats with brain tumor xenografts.</a> Anesthesia & Analgesia. 88, 559-559 (1999).</li>
<li>Nilaver, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Delivery+of+herpesvirus+and+adenovirus+to+nude+rat+intracerebral+tumors+after+osmotic+blood-brain+barrier+disruption%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Delivery of herpesvirus and adenovirus to nude rat intracerebral tumors after osmotic blood-brain barrier disruption.</a> Proceedings of the National Academy of Sciences. 92, 9829(1995).</li>
<li>Fortin, D., McCormick, C. I., Remsen, L. G., Nixon, R., Neuwelt, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unexpected+neurotoxicity+of+etoposide+phosphate+administered+in+combination+with+other+chemotherapeutic+agents+after+blood-brain+barrier+modification+to+enhance+delivery%2C+using+propofol+for+general+anesthesia%2C+in+a+rat+model%5BTitle%5D)+AND+%22Neurosurgery%22%5BJournal%5D)">Unexpected neurotoxicity of etoposide phosphate administered in combination with other chemotherapeutic agents after blood-brain barrier modification to enhance delivery, using propofol for general anesthesia, in a rat model.</a> Neurosurgery. 47, 199(2000).</li>
<li>Neuwelt, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+CNS+lymphoma+treated+with+osmotic+blood-brain+barrier+disruption%3A+prolonged+survival+and+preservation+of+cognitive+function%5BTitle%5D)+AND+%22Journal+of+clinical+oncology%22%5BJournal%5D)">Primary CNS lymphoma treated with osmotic blood-brain barrier disruption: prolonged survival and preservation of cognitive function.</a> Journal of clinical oncology. 9, 1580(1991).</li>
<li>Remsen, L. G., Trail, P. A., Hellström, I., Hellström, K. E., Neuwelt, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+delivery+improves+the+efficacy+of+a+tumor-specific+doxorubicin+immunoconjugate+in+a+human+brain+tumor+xenograft+model%5BTitle%5D)+AND+%22Neurosurgery%22%5BJournal%5D)">Enhanced delivery improves the efficacy of a tumor-specific doxorubicin immunoconjugate in a human brain tumor xenograft model.</a> Neurosurgery. 46, 704(2000).</li>
<li>Neuwelt, E. A., Pagel, M. A., Kraemer, D. F., Peterson, D. R., Muldoon, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bone+marrow+chemoprotection+without+compromise+of+chemotherapy+efficacy+in+a+rat+brain+tumor+model%5BTitle%5D)+AND+%22Journal+of+Pharmacology+and+Experimental+Therapeutics%22%5BJournal%5D)">Bone marrow chemoprotection without compromise of chemotherapy efficacy in a rat brain tumor model.</a> Journal of Pharmacology and Experimental Therapeutics. 309, 594-599 (2004).</li>
</ol>

No conflicts of interest declared.

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...

<table><tbody><tr><td>Long Evans rat</td><td>Harlan Laboratories</td><td></td><td>210-250 g, male</td></tr><tr><td>PE 50 Tubing</td><td>Beckton-Dickinson</td><td></td><td></td></tr><tr><td>18 gauge x 2.5&quot; IV catheter</td><td>Terumo</td><td></td><td>For ET tube</td></tr><tr><td>30&quot; IV extension sets</td><td>Abbott</td><td></td><td></td></tr><tr><td>26 gauge veterinary IV catheter</td><td>Monoject</td><td></td><td></td></tr><tr><td>Evans blue dye</td><td>Sigma</td><td>E2129</td><td></td></tr><tr><td>Bipolar</td><td>Codman</td><td></td><td></td></tr><tr><td>Filter, 5 &amp;mu;m</td><td>Braun</td><td></td><td></td></tr></tbody></table>

rat model of blood-brain barrier disruption to allow targeted neurovascular therapeutics

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

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.

<img alt="Brain hemispheres, A and B, visual comparison, dye uptake experiment, anatomical study." src="/files/ftp_upload/50019/50019fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
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...

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 ...

The overall goal of this procedure is to disrupt the blood-brain barrier. This is accomplished by first placing the animal on IV anesthesia with an endotracheal airway. Next, the bifurcation of the common carotid artery is exposed.Then a catheter is inserted into the external carotid artery. Finally, mannitol is administered. Ultimately, results can be obtained that show blood-brain barrier disruption through extravasation of Evan's Blue Dye.The main advantage of this technique over existing methods of intra arterial therapy is it allows drugs to enter the brain, which do not ordinarily cross the blood-brain barrier under physiological conditions. This has implications in the field of neuroscience because it allows us to test new therapies which have poor ability to cross the blood-brain barrier. Implications of this technique extend to neuro-oncology cause many chemotherapeutic agents do not cross the blood-brain barrier.Well, Though this method provide us insight over new oncology, it can also provide us information on other neurological dis disorders treated by drugs with poor blood brain barrier permeability. We hope that the visual demonstration of this method will allow researchers and familiar with animal surgery to use this technique in their labs. To begin, prepare a solution of 25%mannitol and verify that there are no crystals.If crystals are present, dissolve them by placing the vial in an 80 degree Celsius water bath and agitate. Periodically record the mass of the animal and prepare two 12 cc syringes of propofol to be delivered at a rate of 0.1 milliliters per minute. After anesthetizing the animal in an induction chamber, place it on a disposable towel and shave its neck and inner thighs.Place the animal back in the induction chamber for three minutes. To prepare for intubation to intubate the rat, begin by preparing an endotracheal tube with an IV catheter. The tube must be large enough to allow adequate airflow, but small enough to fit into the trachea without causing damage.To make a stylus, pull the needle out of the plastic catheter and carefully break the needle. Approximately one centimeter from its sharp tip, put the needle back into the catheter. Next, hang the rat by its teeth on an angled surface, shine a light directly against the animal's neck lined up between its ears.Next, insert a small spatula into the oropharynx. Approximately three centimeters. Lift upward while gently pulling the tongue out of the mouth.Visualize the opening of the trachea as a small hole that opens and closes. With each breath, gently insert the catheter into the hole, making sure not to force it into the cartilage of the sensitive trachea. Set the ventilator to one liter per minute and 60 BPM and then isof fluorine to 2%Then connect the catheter to the ventilator and tape the tube to the operating surface.Verify that the animal is anesthetized by performing a toe pinch. Secure the route by taping all four limbs in the tail to the operating surface. Scrub the shaved neck and groin sites with chlorhexidine soap three times.Then scrub with chlorhexidine. Rinse three times. Attach a syringe of propofol to an IV extension set and mount the syringe on the infusion pump.Next, using a scalpel cut through the skin of the shaved thigh without cutting the muscle. Then use forceps to open the incision and to locate the femoral vein and pull away the tissue over the artery to expose the vessel. Insert a 26 G mono eject veterinary IV catheter into the vein.Once the needle punctures the wall of the vein, it'll become easier to push. Retract the needle and push the catheter into the vessel three to five minutes. After beginning the propofol infusion, switch the anesthesia gas to a mixture of 50%O2 and 50%N two O and shut off the isof fluorine.To expose the bifurcation of the common carotid artery, begin by using a scalpel blade to make a midline incision at the neck. Cut through the superficial facia along the midline to reach the sternal hyoid muscle over the trachea. Move the tissue away laterally to visualize a group of three muscles, the sternal hyoid, the di gastric, and the sternal mastoid that formed the rat carotid triangle with forceps or scissors.Separate the fascia between the muscles of the carotid triangle to expose the carotid artery. Use bipolar cautery to divide minor vessels along with gauze and cotton swabs To control bleeding, use retractors to hold the muscles apart with forceps. Move the tissue off of the carotid and find the bifurcation.Expose the occipital artery just distal to the bifurcation. Cauterize it between forceps and use an electrocautery to separate it with forceps. Fully expose the carotid bifurcation and as much of the external carotid artery or ECA as possible.Expose the superior thyroid artery branching off the ECA distal to the occipital artery. Cauterize it between forceps and separate it to catheterize the ECA. Place two lengths of four oh silk under it.Use a hemostat and grab one length of suture and retract the ECA back coly with the other suture. Tie it with the surgeon's knot as far cranial as possible, distal to the bifurcation and proximal to the permanent ligature. Loosely tie another surgeon's knot to relax the artery.Irrigate it with 1%lidocaine, fill the catheter with heparinized saline. Next, place a temporary vessel clip at an angle as close as possible to the bifurcation. Then slide the loose knot toward the clip using micro scissors.Make a small cut in the ECA just proximal to the ligature. If the clip and ligature are secure, there should be no bleeding using forceps. To control the ECA walls, feed the beveled end of the catheter into the vessel with the loose surgeon's knot.Secure the catheter. Tie the artery around the catheter, then remove the clip and use a cotton swab to clean the area. Advance the catheter about one millimeter distal to the bifurcation.Use forceps to hold the artery proximal to the knot to ensure the vessel is immobilized. To administer mannitol. Fill an eight inch IV extension set and a six milliliter syringe with mannitol without creating any air bubbles.Store it at 37 degrees Celsius to visualize the blood-brain barrier disruption, or BBBD administer two milligrams per kilogram of 2%Evans Blue through a new IV site on the leg contralateral to the propofol infusion. Attach the extension set to a five micron filter and fill it with minol to remove air bubbles. Then attach it to the stop cock without creating bubbles.Administer mannitol at 0.09 milliliters per second For 25 seconds, watch for the mannitol to reach equilibrium with the flow in the common carotid at a point just proximal to the internal carotid artery, it should appear to pulse with the rat's heart rate. Ensure that the mannitol does not flow toward the heart, which indicates the flow has overcome the rat's blood pressure and can cause serious complications. Disruption of the blood-brain barrier on one side of the brain allows Evan's blue to enter and stain the disrupted hemisphere blue, while the non-disruptive hemisphere remains unchanged.Shown here is an example of a brain without blood-brain barrier disruption or BBBD, and here is an example of a brains stained blue after osmotic BBBD, notice that the right hemisphere is stained and that some blue is entered the left hemisphere near the medial longitudinal fissure as a result of mixing of blood flow at the circle of Willis. This technique paves the way for researchers to explore therapies which do not cross the blood-brain barrier in a rat model. After watching this video, you should be able to disrupt the blurred brain barrier by catheterizing the carotid artery and infusing a manitol solution directly into the brain.

rat-model-blood-brain-barrier-disruption-to-allow-targeted

Research

JoVE Journal

Medicine

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

In this video, we describe a comprehensive protocol to establish an in vitro model mimicking the blood-brain tumor barrier. The model holds promise to understand the delivery of therapeutic agents for the treatment of brain tumors.

Blood-Brain Tumor Barrier or BBTB Model: An Adaption of the Blood-Brain Barrier or BBB Model to Evaluate Delivery of Therapeutics to the Tumor

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

Drug targeting to central nervous system tumors is a major challenge. Here we describe a protocol to produce an in vitro mimic of the blood-brain tumor-barrier using murine and/or human cells and discuss their relevance for the predictability of central nervous system tumor targeting in vivo.

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

This protocol describes a method to establish a human blood-brain barrier (BBB) in vitro model. The endothelial cells and pericytes are seeded on each side of an insert filter (blood compartment), and astrocytes are seeded in the bottom well (brain compartment). The model characterized was used for nanoparticles transport experiments.

A Triple Culture Cell System Modeling the Human Blood-Brain Barrier

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

Learn to disrupt the blood-brain barrier in rats using intra-arterial mannitol infusion via external carotid catheterization. This method enables targeted neurovascular drug delivery confirmed by Evan's Blue extravasation.

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

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