We would like to acknowledge the FCCC Laboratory Animal Facility for their support in this project. We thank Wolfe Laboratories, Inc. for their assistance in analyzing paclitaxel levels in plasma and tissue. This work was supported by National Institutes of Health grants K01CA120091 to E.H.B., and CA06927 to Fox Chase Cancer Center.

This protocol has been approved by the Fox Chase Cancer Center Institutional Animal Care and Use Committee and by the Laboratory Animal Facility, and found to be in accordance with institutional guidelines for humane treatment of animals.
1. Preliminary Preparation
<ol>
	<li>Preparation of catheter: Prepare a catheter from a short length of polyethylene tubing, modified to form a thinned, blunted end (Figure 3A). Make multiple catheters in advance and save indefinitely.
	<ol>
		<li>Light a Bunsen burner, and adjust to establish a low steady flame. Hold tubing close to flame to soften polyethylene. When tubing begins to melt, slowly pull apart the two ends to create a thinned section of tubing, approximately 0.25 mm OD.</li>
		<li>Cut a beveled end approximately 0.75 cm along thin section. This ensures enough tubing to be secured in artery, without creating an overly long catheter. 
		NOTE: A long thin end on the catheter has a propensity to clog. An overly long end may also hold enough liquid to significantly change the catheter&#8217;s liquid volume capacity.</li>
		<li>Blunt the beveled end by passing quickly through the flame &#8212; when properly heated, the end becomes slightly rounded and enlarged. The tip hooks back slightly, which helps anchor the tubing while inserting into the artery.</li>
		<li>Cut the tubing 6.0 cm from the point where it begins to thin. This makes a very manageable catheter, that is long enough to thread through to exit from the neck and to hold and work with comfortably, but short enough to prevent the mouse from gnawing on excess tubing or requiring too much extra infusion volume to clear initial saline.</li>
		<li>Prepare a syringe of approximately 0.2 ml heparin solution, topped with a blunted needle. Insert needle into the wide end of the catheter (Figure 3B). Fill catheter with heparin, being careful to ensure that there are no bubbles in the tubing. Set the heparin syringe and catheter aside on a sterile area.&#160;Sterilize catheters by gamma irradiation, by&#160; placing catheter(s) in a petri dish, and exposing to 20 Gy of gamma irradiation. If you do not have access to an gamma radiation source, check with your animal facility to investigate other means of sterilization, such as gas- or chemical-sterilization.&#160; Do not autoclave, as polyethylene cannot be heat sterilized.</li>
	</ol>
	</li>
	<li>Creation of a saline lead (Figure 3B).
	<ol>
		<li>Prepare a second syringe of approximately 0.5 ml sterile saline, being careful to ensure a bubble-free line.</li>
		<li>Cut a second piece of tubing, approximately 15 cm, and slip onto the blunted syringe needle. Attach a connector port to the free end of the tubing.</li>
		<li>Test that flow of saline is unobstructed by smoothly advancing a small volume of saline through the lead. Use this saline lead after catheter insertion, to check the flow through the catheter, and to flush the line of blood. Set the saline syringe aside on a sterile area.</li>
	</ol>
	</li>
	<li>Prior to surgery, sterilize equipment by autoclaving, or alternatively by gas sterilization or glass bead sterilization.</li>
	<li>Prepare sterile surgical area.
	<ol>
		<li>Wipe bench and microscope surfaces with disinfectant such as 70% ethanol or chlorine dioxide. Cover bench and microscope base with a clean disposable, absorbent pad.</li>
		<li>Prepare a surgical board by covering with two layers of clean, absorbent paper, securely attached with adhesive tape.</li>
	</ol>
	</li>
	<li>Lay out all surgical supplies (as cataloged in Materials List) so that they are readily accessible.
	<ol>
		<li>Cut three lengths of sterile suture, 8 cm each, and set aside with other supplies. Place a port where it will be easily available (Figure 3B).</li>
	</ol>
	</li>
</ol>
2. Surgery
<ol>
	<li>Preparation of the animal
	<ol>
		<li>Anesthetize mouse by exposure to 2-3% isoflurane in anesthesia chamber connected to precision vaporizer.&#160; Withdraw mouse from the chamber, and shave hair from the mouse&#8217;s neck / upper torso, and below the right ear (site of catheter exit). Administer veterinary petrolatum ophthalmic ointment to eyes to prevent dryness while under anesthesia. Ensure the animal does not awaken during surgical preparations by allowing mouse sufficient time in inhalation chamber before prep (at least two min), or by administering isoflurane to&#160; mouse by means of a nose cone during prep. Return mouse to anesthesia chamber.</li>
		<li>When mouse is sufficiently inert, move to sterile surgical area, place anesthesia nose cone over the mouse&#8217;s nose and mouth, and divert the flow of isoflurane to nose cone. Confirm proper anesthetization by pinching paw with forceps; when the mouse shows no reaction, proceed to next step.</li>
		<li>Position the mouse on its back, with the head facing toward the investigator. Secure the ears, fore-paws and hind-paws to surgical board with adhesive tape or other restraining device to keep the mouse steady. Clean the incision area with povidone-iodine and 70% ethanol.</li>
	</ol>
	</li>
	<li>Isolation of carotid artery
	<ol>
		<li>Make a 1 cm longitudinal cut slightly to the right of the midline of the neck. Use forceps to separate fat and muscle to expose the trachea (Figure 4A). Locate the carotid artery running parallel to the trachea (Figure 4B).</li>
		<li>Carefully use forceps to separate fascia overlying the artery (Figure 4C). Lightly pull the vagus nerve aside from the carotid, and insert forceps into the space between. Gently open forceps to create a gap in the fascia, and carefully pull away the nerve from the artery, from the fork in the artery near the larynx (anterior end), up (posterior) as far as possible (at least 3 mm) (Figure 4D).</li>
		<li>Clear away any remaining fascia until the artery is well isolated (Figure 4E). Add a drop of saline to the surgery area occasionally to keep tissue moist, and thus less brittle and less prone to tearing randomly.</li>
	</ol>
	</li>
	<li>Suture placement and artery preparation for catheter insertion (Figures 4, 5).
	<ol>
		<li>Use forceps to draw a silk suture thread under the artery. Tie a secure knot to close off the artery as far toward the anterior as possible (Figure 4F).</li>
		<li>Draw a second thread under the artery. Tie a retractable knot to temporarily close off the artery as far toward the posterior as possible (Figure 4F).</li>
		<li>Draw a third thread under the artery. Tie a very loose knot between the first two sutures, to be used to quickly secure the catheter after placement (Figure 4G).</li>
		<li>Hold ends of all sutures out of the way by wetting them with a bit of 70% ethanol.</li>
	</ol>
	</li>
	<li>Catheter insertion
	<ol>
		<li>With the suture, grab the lower knot to pull the artery slightly taut. Nick the artery above, but very close to, the anterior suture (Figure 4H). Be careful not to cut too deeply, but check the slit to make sure the opening is unobstructed.</li>
		<li>Remove heparin-filled catheter from syringe needle, trying to avoid creating large pockets of air at either end. Manipulate the catheter to position bevel at a comfortable angle, generally downward, and slightly to the right (for right-handers).</li>
		<li>While holding on to the suture to keep the artery remaining slightly taut, gently insert catheter into slit (Figure 4I). Use the forceps to hold the anterior suture and pull the artery down over catheter (pushing up excessively with the catheter may cause the beveled end to puncture the artery). Carefully release the catheter and anterior suture.</li>
	</ol>
	</li>
	<li>Securing of catheter and initiation of blood flow (Figures 4J, 5B).
	<ol>
		<li>Secure the catheter by tightening the knot of the middle suture, close to the entrance of the catheter into the artery. Make a tight triple knot, but be sure not to pull so tight as to obstruct flow through the catheter. Further secure the catheter by tying it down with the anterior suture, below the entrance to the artery.</li>
		<li>Attach the saline lead to the catheter by means of the connector plug, again trying to avoid introducing air bubbles into the line.</li>
		<li>Grasp the ends of the posterior suture and gently pull to release the knot. Maneuver the suture down the artery, over the end of the catheter (do not remove the thread). Blood should flow into the catheter; if there is no blood flow, gently wiggle catheter to attempt to remove the constriction.</li>
		<li>When flow appears unobstructed, use the last thread (from the posterior suture) to tie an additional knot, slightly above the middle suture.</li>
	</ol>
	</li>
	<li>Temporary sealing of catheter. Flush the catheter of blood, then use a hemostat to clamp the end of the catheter close to the connector plug. Remove the connector and replace it with the port plug to seal off the end of the catheter, and remove the hemostat.</li>
	<li>Repositioning catheter to exit from back of neck.
	<ol>
		<li>With forceps in each hand, use one pair of forceps to hold on to the catheter just below the anterior suture, and with the other, press a kink into the catheter so it will easily bend to the side. Repeat to create a second kink. This allows the free end of the catheter to be pulled toward the back of the mouse&#8217;s head, without forcing the tip of the catheter to turn laterally against the wall of the artery.</li>
		<li>Turn the mouse onto its (left) side, keeping the nose cone positioned over the mouth and nose, and clean the incision area with 70% ethanol and povidone-iodine.&#160;Make a small incision (approximately 4 mm) below and behind the right ear.</li>
		<li>Use forceps to hold the open flap of skin, while working the blunt hollow probe under skin to create a channel through the cheek, to the cavity at the neck. It is advisable to bring the probe around the salivary gland, instead of trying to go between the gland and the skin. Use forceps to carefully free a space for the probe to exit.</li>
		<li>Thread port plug/catheter through probe to exit in neck. Do not pull too hard; be sure the catheter is not crushing or constricting blood vessels or organs.</li>
	</ol>
	</li>
	<li>Closure and recovery. Administer topical analgesic (e.g. bupivacaine) to the shoulder incision, and cover the wound with water-proof, surgical adhesive tape. Apply a second piece of adhesive tape to further secure the catheter.
	<ol>
		<li>Administer topical analgesic to chest incision, and close wound with silk or staples.</li>
		<li>Remove mouse from anesthesia, and allow animal to recover in a clean, warmed space (place cage on top of a heating pad or under a heat lamp), for at least 30 min.</li>
	</ol>
	</li>
</ol>
3. Infusion
<ol>
	<li>Prepare aliquots of 5 mg/ml solution of paclitaxel / methanol.
	<ol>
		<li>Measure 50 mg of paclitaxel into a 15 ml centrifuge tube. Add 10 ml of sterile methanol. Cap tube. Rotate by hand or on a rotating shaker at room temperature until powder is dissolved.</li>
		<li>Aliquot 500 &#181;l of solution into 20 small, freezer safe tubes, and store at -20 &#176;C.</li>
		<li>Thaw individual aliquot at room temperature or in 37 &#176;C water bath, immediately before infusion.</li>
	</ol>
	</li>
	<li>Prepare the infusion pump (Figure 6).
	<ol>
		<li>Cut a long length of polyethylene tubing of approximately 40 cm. Attach a blunted needle to one end, and a connector port to the other.</li>
		<li>Draw up drug into a syringe with a known inner-diameter (most programmable pumps will require the diameter of the syringe barrel, in order to calculate the speed of the pump arm). Attach the needle to the syringe and load the drug through the needle and tubing.</li>
		<li>Situate the syringe into the pump according to the manufacturer&#8217;s directions. Prime the pump so that the drug is flowing smoothly out the connector plug, and it is ready for infusion.</li>
	</ol>
	</li>
	<li>Attach mouse to pump.
	<ol>
		<li>Hold the mouse steady and use the hemostat to clamp catheter, close to the port plug. Remove plug and replace with the connector attached to the syringe and tubing. 
		NOTE: Blood may start to flow back through tubing.</li>
		<li>Quickly administer a fast pump to clear the volume of the catheter (calculated empirically through observing the volume of 6 cm of tubing), then immediately switch to the desired infusion rate.</li>
	</ol>
	</li>
	<li>Continue Paclitaxel infusion for three hour time course.
	<ol>
		<li>Monitor tubing occasionally to check for leaks at junctions as this is often a sign of a blockage in the flow to the mouse. Watch the mouse for expected or unexpected reactions to infusion (lethargy or hyperactivity, signs of discomfort).</li>
		<li>Depending on the length and nature of the infusion, the mouse might not eat or drink, but be sure to provide access to food and water according to the institution&#8217;s established policy. Be aware of the potential for dehydrating the mouse through collection of large quantities of blood.</li>
		<li>Continue to keep the cage warm with a heating pad or lamp, unless the mouse appears to desire to stay away from the heat. If animal is not euthanized within several hr after surgery, implement plan for post-surgical treatment of animal, including sterile housing conditions and treatment for post-surgical pain.</li>
		<li>Keep a close watch on the mouse, especially in the first few min, to ensure it does not pull out the tubing through hyperactivity, or irritation from tubing (which may be a sign of a poor insertion). If the mouse is not over-active, constant monitoring may not be necessary, but check the mouse routinely to be sure the animal does not get tangled in the tubing. Harness and tether system are available commercially, but their use is beyond the scope of this protocol.</li>
	</ol>
	</li>
	<li>Collection of samples.
	<ol>
		<li>Collect blood samples at regular intervals by submandibular or retro-orbital bleeds (If your protocol does not make use of mammary tumor models, consider collection of blood via a jugular catheter inserted at the same time as the carotid catheter).&#160; Be careful not to pull on the infusion line. If collecting by retro-orbital bleeds, lightly anesthetize the mouse with an inhalational anesthetic (e.g. methoxyflurane) so one need not grab by the scruff to secure the mouse.</li>
		<li>Spin blood in hemocrit centrifuge to separate blood cells from plasma. Use a file or diamond-tip pen to score tube at face of phase separation. Break tube and collect plasma only, into small, freezer-safe tube. Store at -80 &#176;C until analysis. 
		NOTE: If a hemocrit centrifuge is not available, transfer blood sample into micro-centrifuge tube, and spin in a micro-centrifuge at high speed to separate blood cells from plasma. Collect plasma into a second tube, and store at -80 &#176;C.</li>
		<li>Euthanize mouse by CO2 asphyxiation. Collect tissue (about 20 &#8211; 50 mg) from organs of interest and flash freeze in liquid nitrogen. Store at -80 &#176;C until analysis.</li>
	</ol>
	</li>
</ol>
4. Sample Analysis
NOTE: All samples for this protocol were analyzed through an outside laboratory by liquid chromatography-tandem mass spectrometry (LC-MS/MS), who calculated the paclitaxel concentrations as follows:
<ol>
	<li>Extract paclitaxel from samples. Homogenize tissue sample in 0.1% acetic acid, 50% methanol prior to extraction. Extract paclitaxel by liquid/liquid extraction, using methyl tert-butyl ether (MTBE) fortified with an internal analogue (docetaxel) standard. Remove MTBE and dry samples. Resuspend in 50% acetonitrile, 0.1% acetic acid solution.</li>
	<li>Prepare calibration standards. Add a known concentration of paclitaxel to appropriate C57BL/6 matrix to obtain a final range of standards (from 1 to 20,000 ng/ml for plasma samples, from 0.1 to 5,000 ng/ml for tissue samples). Extract standards in duplicate, using the same method as for the study samples above. Measure paclitaxel peak in samples by HPLC/MS/MS utilizing electrospray ionization.</li>
	<li>Calculate concentration using the area ratio of paclitaxel to internal standard. Use calibration standards to create a standard curve, and interpolated study samples by fit on the curve. Normalize concentration of study samples by the starting weight of sample before homogenization.</li>
</ol>

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	<li>Gallo, J. M., Li, S., Guo, P., Ma Reed, K., J, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+P-Glycoprotein+on+Paclitaxel+Brain+and+Brain+Tumor+Distribution+in+Mice.">The Effect of P-Glycoprotein on Paclitaxel Brain and Brain Tumor Distribution in Mice.</a> Cancer Research. 63 (16), 5114-5117 (2003).</li><li>Sonnichen, D. S., Relling, M. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+Animal+Usage+by+Serial+Bleeding+of+Mice+for+Pharmacokinetic+Studies:+Application+of+Robotic+Sample+Preparation+and+Fast+Liquid+Chromatography+&#8211;+Mass+Spectrometry.">Reduction of Animal Usage by Serial Bleeding of Mice for Pharmacokinetic Studies: Application of Robotic Sample Preparation and Fast Liquid Chromatography &#8211; Mass Spectrometry.</a> Journal of Chromatography B: Biomedical Sciences and Applications. 754 (1), 245-251 (2001).</li><li>Berndt, K., Vogel, J., Buehler, C., Vogt, P., Born, W., Fuchs, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+repeated+drug+infusion+into+the+femoral+artery+of+mice.">A new method for repeated drug infusion into the femoral artery of mice.</a> J. Am. Assoc. Lab Animal Sci. 51 (6), 825-831 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Squibb+&amp;+Sons,+L.L.C..">Squibb &amp; Sons, L.L.C..</a> Drug Information for TAXOL (Paclitaxel) Injection. , (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abraxis+BioScience+L.L.C.">Abraxis BioScience L.L.C.</a> Full Prescribing Information ABRAXANE for Injectable suspension (paclitaxel protein-bound particles for injectable suspension) (albumin-bound). , (2013).</li><li>Fergusson, G., Ethier, M., Zarrouki, B., Font&#233;s, G., Poitout, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Model+of+Chronic+Nutrient+Infusion+in+the+Rat.">A Model of Chronic Nutrient Infusion in the Rat.</a> J. Vis. 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The authors have nothing to disclose.

Carotid artery infusion is a significant technique in this study of paclitaxel pharmacokinetics. Carotid artery infusion is a method to quickly distribute drug throughout the circulatory system14. The 3 hr infusion is a closer mimic of clinical administration of drugs such as taxanes than bolus injections. The surgery can be reliably performed by a single individual, surgery time is relatively short, and success rates are &#62;75%. After samples are collected, they must be analyzed by the appropriate methods. ...

Drug infusion through the carotid can be performed reliably and reproducibly by optimizing equipment and technique. The procedure is not intricate, although it does require fine control and attention to detail. Superior care and dexterity are needed to isolate the carotid artery and insert the catheter, which can generally be acquired through practice. Surgery by an experienced technician should not exceed one hour. After successful surgery, the mouse should appear normal and healthy (although the mouse may react to the actual drug infusion), and drug(s) may be administered in a controlled, uniform continuous dosage. Blood samples must be taken from a site other than the carotid artery; retro-orbital bleeds proved easy to collect and satisfactory for analysis of drug concentrations.
Catheters of optimum size and shape are an invaluable asset in performing a successful infusion11. We found the catheters available commercially often to be too large and/or too flexible to allow for convenient access to the mouse carotid artery. It proved preferable to fashion catheters from the polyethylene tubing used to connect the mouse to the infusion syringe. Thus, all the tubing, connectors and needles were of consistent dimensions, which simplified infusion assembly. Using this technique, it is not necessary to push the tip of the catheter into the artery past the point where it is still visible, and blood flow to the carotid artery is not restored until after the catheter is initially secured. This reduces the hazards of puncturing the artery or of having the catheter pushed out by the high pressure of blood flow. The catheter design herein does not incorporate a &#8220;bump&#8221; to hold it in place, so securing the catheter well with sutures and surgical tape is a priority.
Infusions may be preferable to the common i.v. bolus injections, as a better mimic of clinical administration of drugs such as taxanes3, 12, 13. The technique described here was originally developed to allow infusion into mouse models wherein access to the jugular or femoral vein was precluded by mammary tumor growth and/or excessive vascularization of the insertion area. This method may often be appropriate even in tumor-free mice: although isolating and catheterizing the carotid is slightly more invasive, we found it preferable to the jugular, because the propensity of the jugular wall to rip resulted in more failed insertions and failures to complete the 3 hr time course.
While the results shown here are from C57BL/6J (in-house-bred) mice, we have used this technique to successfully infuse paclitaxel into several strains of mice, including FVB and mixed-strains, to follow the pharmacokinetics in mouse models transgenically manipulated to down-regulate cellular transporter functions. The blood and tissue samples collected showed expected levels of paclitaxel, in the range of the levels seen after jugular infusions1. This technique may be expected to work equally well in other mouse models and with other infusion solutions.

<table border="0" cellpadding="0" cellspacing="0" width="846">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:293px;">Table of specific reagents and equipment:</td>
			<td style="width:185px;">Source:</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:259px;">Comments</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">Polyethylene tubing 0.024&#8221;OD X 0.011&#8221;ID&#160;</td>
			<td style="width:185px;">Braintree Scientific, Inc.</td>
			<td style="width:111px;">PE10</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">Stainless steel port plug (30 gauge)</td>
			<td style="width:185px;">Braintree Scientific, Inc.</td>
			<td style="width:111px;">NB-30</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">2 Stainless steel connector plugs (28 gauge)</td>
			<td style="width:185px;">Braintree Scientific, Inc.</td>
			<td style="width:111px;">PP-28</td>
			<td style="width:259px;">Slightly larger than PE tubing ID, to fit snugly and keep a tight seal.</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">3 Blunted needles (30 gauge)</td>
			<td style="width:185px;">Braintree Scientific, Inc.</td>
			<td style="width:111px;">C-30</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">3 1cc syringes</td>
			<td style="width:185px;">Becton, Dickinson and Co.</td>
			<td style="width:111px;">309659</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">Sterile 0.9% Saline solution</td>
			<td style="width:185px;">Hospira</td>
			<td style="width:111px;">0409-7984-37</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">Cath-Loc HGS Heparin/Glycerol Solution&#160;</td>
			<td style="width:185px;">Braintree Scientific, Inc.</td>
			<td style="width:111px;">HGS</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">Silk suture</td>
			<td style="width:185px;">Braintree Scientific, Inc.</td>
			<td style="width:111px;">SUT-S&#160;113</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">Vanna Scissors (micro-scissors)</td>
			<td style="width:185px;">World Precision Instruments</td>
			<td style="width:111px;">14122</td>
			<td style="width:259px;">This model has a curved tip, but straight-tip scissors work as well.</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">Hartman Mesquito Hemostatic Forceps</td>
			<td style="width:185px;">World Precision Instruments</td>
			<td style="width:111px;">501705</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">Betadine Swabsticks</td>
			<td style="width:185px;">Perdue Products L.P.</td>
			<td style="width:111px;">BSWS1S</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">Bupivacaine</td>
			<td style="width:185px;">Hospira</td>
			<td style="width:111px;">0409-1160-01</td>
			<td style="width:259px;">May be replaced with Lidocaine, or similar local anesthesia.</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">Paclitaxel</td>
			<td style="width:185px;">LC Laboratories</td>
			<td style="width:111px;">P-9600</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">Methanol</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:111px;">32213</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">Micro-Hematocrit Capillary Tubes, Heparinized</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:111px;">22-362-566</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">&#160;Capillary Tube Sealant&#160;</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:111px;">02-678</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:293px;">C57BL/6J mice</td>
			<td style="width:185px;">Fox Chase Cancer Center, Laboratory Animal Facility in-house-bred</td>
			<td style="width:111px;"></td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:293px;">API 4000 Q-Trap mass spetrometer</td>
			<td style="width:185px;">Applied Biosystems</td>
			<td style="width:111px;"></td>
			<td style="width:259px;"></td>
		</tr>
	</tbody>
</table>

carotid artery infusions for pharmacokinetic and pharmacodynamic analysis of taxanes in mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

Paclitaxel distribution follows predictable patterns during a 3 hr dosing regimen of a 15 min high-speed infusion, followed by a 165 min low-speed infusion.
Figure 1 shows a comparison of jugular vein-infusion plasma paclitaxel concentrations and carotid artery-infusions. The paclitaxel concentrations drop quickly in the first 15 min following an initial high volume infusion, and then level off over the next 150 min. By comparison, paclitaxel levels in a poor infusion start of...

Watch this Scientific Journal Video about Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice at JoVE.com

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

carotid-artery-infusions-for-pharmacokinetic-pharmacodynamic-analysis

Research

JoVE Journal

Medicine

22.7K vistas.  Fox Chase Cancer Center.  Este método fue desarrollado con el objetivo de ofrecer una solución de fármaco constante a través de la arteria carótida, para evaluar la farmacocinética de nuevos fármacos en modelos de ratón. 

Video: Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...