Name: catheterization of the carotid artery and jugular vein to perform hemodynamic measures, infusions and blood sampling in a conscious rat model
Uploaded: 2015-01-30T13:00:00
Duration: 9 min 5 s
Description: Watch this Scientific Journal Video about Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model at JoVE.com

Intramural NIH program supported the development of this model. Publication support was provided by Harvard Apparatus Inc. and ADInstruments Inc. The work by the authors was done as part of US government&#8211;funded research; however, the opinions expressed are not necessarily those of the National Institutes of Health.

The procedures described below were performed as part of a protocol approved by the Animal Care and Use Committee of the Clinical Center at the National Institutes of Health.
1. Preparation for Surgery
<ol>
	<li>Gas sterilize catheters and instruments.</li>
</ol>
2. Prepare the Catheters
<ol>
	<li>Flush the catheters with heparinized glycerol (250 IU heparin/1 ml glycerol) to ensure patency and avoid leakage. Plug end of the catheter with a metal stopper (20 ga, 15 mm) to retain heparinized glycerol.</li>
</ol>
3. Prepare Aseptic Workstation
<ol>
	<li>Use 70% alcohol to spray the workstation and instrument trays. Use a sterile drape to cover the operating table and instrument trays and place instruments on the tray.</li>
</ol>
4. Prepare Rats for Surgery
<ol>
	<li>Place rat individually in a Lucite chamber and expose to an anesthetic; isoflurane (3-5%) mixed with oxygen until unconscious. Remove rat from the chamber and shave fur from anterior and posterior areas of the neck. Apply lubricant ointment to eyes.</li>
</ol>
5. Catheter Implantation
<ol>
	<li>Place anesthetized rat (confirmed by toe pinch) onto a heated surgical table in the ventral position; place its nose into the nose cone to maintain anesthesia with spontaneous respirations (isoflurane; 3.5%). Gently scrub the surgical area 3 times alternating use of Betadine and 70% alcohol. Make a 0.5 cm midline skin incision between the scapulae using a surgical scissor.</li>
	<li>Reposition rat in the dorsal position; gently restrain the legs to each side of the table using rubber bands; maintain anesthesia. Place two rolled sterile 4x4 gauze under neck to slightly hyperextend for better exposure. Make a 2 cm ventral cervical skin incision right of the midline of the neck at the level of the clavicle using a scalpel.</li>
</ol>
6. Right Jugular Vein Catheterization
<ol>
	<li>Using a hemostat, bluntly dissect the right jugular vein, separate out the salivary and lymphatic tissues to visualize and isolate a 5 mm section of the vessel. Using 4-0 silk suture, place a loose tie on both cranial and caudal ends of the vessel to maximize the exposure of the vessel. Using a micro surgical scissor make an incision large enough to pass the catheter, in line with the vessel between the two ligatures and tie the cranial ligature around the vessel.</li>
	<li>Insert the venous catheter into the vessel towards the heart with the assistance of the micro dissecting hook and forceps and advance the catheter until all of the PU 3F segment is in the vessel. Use the ligatures at the cranial and caudal ends to secure the catheter to the vessel.</li>
</ol>
7. Left Carotid Artery Catheterization
<ol>
	<li>Using a hemostat, bluntly dissect the omohyoid muscle longitudinally to expose the left carotid artery and isolate a 5mm section of the vessel. Ensure the vagus nerve (white in color) is completely separated from the artery. Take care not to shred or break the nerve.</li>
	<li>Using 4-0 silk suture, place a loose tie on the caudal end of the vessel, tie off the cranial end of the vessel and place a bulldog clamp caudally above the suture to stop the blood flow following the incision. Using a micro surgical scissor, make an incision, large enough to pass the catheter, in line with the vessel between the two ligatures. Insert the arterial catheter towards the heart with the assistance of the micro dissecting hook and forceps.</li>
	<li>Use a smooth needle holder without lock to hold the portion of the catheter inside the vessel tight before removing the bulldog clamp. Advance the catheter with a pair of forceps while loosening needle holder slowly until the entire PU 2F segment of catheter is in the vessel. Tie the loose caudal ligature around the catheter and vessel to secure, but not so tight as to occlude, the catheter.</li>
</ol>
8. Using a Straight Hemostat, Tunnel a 5 cm Tube back Subcutaneously behind the Ear and through the Incision between the Scapulae. Exteriorize the Catheters through the Tube and Remove the Tube.
9. Close the Ventral Incision with Three Stainless Steel Wound Clips, and the Dorsal Incision with 4-0 Silk Sutures to Secure the Exteriorized Catheters in Place.
10. Post-surgical Monitor and Care
<ol>
	<li>Following catheterization, terminate the anesthesia and recover the rat in lateral decubitus position in a cage with cellulose bedding. Observe the rats at 2 hr intervals for at least 4 hr or until showing no signs of pain and then once daily. Give Ketoprofen [5 mg/kg, subcutaneous injection (SQ)] immediately post procedure and every 12 hr if the rat shows signs of pain or distress. In our experience, no rats have required additional analgesia for pain following the initial injection. Characteristics observed that would elicit consideration for additional doses of analgesia include but not limited to; abnormal posture; increase/decrease in respiratory rate, unthrifty/ungroomed, infection/inflammation of the incision site, eye discharge, piloerection (&#8220;spiked haircoat&#8221;), reduction in activity in response to audible or tactile stimuli. 
	NOTE: Rats recovered for 72 hr that gain &#62;10 g from pre-catheterization weight will be enrolled in the study.</li>
</ol>
11. Connection to Hemodynamic System
<ol>
	<li>Following recovery, enroll healthy rats (weight gain &#62; 10g) in the study. Place rats in Lucite chamber and anesthetize as previously described.</li>
	<li>Place sterile drape on surgical table with sterile hemostat, micro forceps, 1 ml syringes with blunt needle attached. 
	NOTE: The tips of the hemostat should be covered with rubber to avoid damage to the catheter. Place anesthetized rat on a surgical table in the ventral position and place nose into the nose cone to maintain anesthesia (3-5%) with spontaneous respirations.</li>
	<li>Clamp arterial and venous catheters using a pair of rubber capped hemostats just below the metal plug pins. Use a pair of forceps to remove the metal pin at the end of the catheter and attach a 1 ml syringe filled with heparin saline with a blunt needle attached. In turn, remove the hemostats and withdraw 0.1 ml to ensure the catheters are patent.</li>
	<li>Re-clamp the catheters, remove the needle and attach this end to a longer catheter (pre-flushed with heparin saline) that&#8217;s connected to a swivel and tethered on top of the shoebox cage. Connect these catheters to the transducers for data acquisition and recording, sampling, or infusion. 
	NOTE: All catheters are protected with coiled spring conduits long enough to allow an animal&#8217;s full range of motion in their cage.</li>
	<li>Following catheter connection, terminate anesthesia and recover the animals in the sternal position in a cage with cellulose bedding. To maintain arterial catheter patency, manually flush the lines with 0.05 ml heparin saline (50 IU/ml) every 1 hr for 24 hr which coincide with the regular assessments of the animals.</li>
</ol>
12. Blood Sampling, Pressure Monitoring and Drug Administration
<ol>
	<li>Attach empty syringe to stopcock connected to arterial catheter and withdraw 0.6 ml inline flush. Attach sample syringe, take sample, and return the 0.6 ml inline flush followed by 0.7 ml heparinized saline flush.</li>
	<li>Use data acquisition hardware and software to measure and record MAP and heart rate continuously.</li>
	<li>Connect the automated drug delivery system (ADDS) to the venous catheter. 
	NOTE: ADDS adjusts the rate of vasopressor infusion (high, low, or 0 dose) based on the MAP detected to maintained pressures within the normal physiologic range.</li>
</ol>

<ol>
	<li>Buckingham, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indwelling+catheters+for+direct+recording+of+arterial+blood+pressure+and+intravenous+injection+of+drugs+in+the+conscious+rat.">Indwelling catheters for direct recording of arterial blood pressure and intravenous injection of drugs in the conscious rat.</a> J Pharm. Pharmacol. 28 (5), 459-461 (1976).</li><li>Buckle, J. W., Nathaniels, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proceedings:+A+dual+catheter+system+for+the+simultaneous+infusion+and+sampling+of+the+vascular+system+of+the+unrestrained+rat.">Proceedings: A dual catheter system for the simultaneous infusion and sampling of the vascular system of the unrestrained rat.</a> J. Physiol. 242 (2), 55P-56P (1974).</li><li>Solomon, S. B., 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=Effective+dosing+of+lipid+A+analogue+E5564+in+rats+depends+on+the+timing+of+treatment+and+the+route+of+Escherichia+coli+infection.">Effective dosing of lipid A analogue E5564 in rats depends on the timing of treatment and the route of Escherichia coli infection.</a> J. Infect. Dis. 193 (5), 634-644 (2006).</li><li>Cui, X., 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=Bacillus+anthracis+cell+wall+produces+injurious+inflammation+but+paradoxically+decreases+the+lethality+of+anthrax+lethal+toxin+in+a+rat+model.">Bacillus anthracis cell wall produces injurious inflammation but paradoxically decreases the lethality of anthrax lethal toxin in a rat model.</a> Intensive Care Med. 36 (1), 148-156 (2010).</li><li>Quezado, Z. M., Natanson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+hemodynamic+abnormalities+and+vasopressor+therapy+in+sepsis+and+septic+shock.">Systemic hemodynamic abnormalities and vasopressor therapy in sepsis and septic shock.</a> Am. J. Kidney Dis. 20 (3), 214-222 (1992).</li><li>Perdue, P. W., Kazarian, K. K., Nevola, J., Law, W. R., Williams, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+local+and+systemic+antibiotics+in+rat+fecal+peritonitis.">The use of local and systemic antibiotics in rat fecal peritonitis.</a> J. Surg. Res. 57 (3), 360-365 (1994).</li><li>Qiu, 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=The+individual+survival+benefits+of+tumor+necrosis+factor+soluble+receptor+and+fluid+administration+are+not+additive+in+a+rat+sepsis+model.">The individual survival benefits of tumor necrosis factor soluble receptor and fluid administration are not additive in a rat sepsis model.</a> Intensive Care Med. 37 (10), 1688-1695 (2011).</li><li>Cui, X., 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=Severity+of+sepsis+alters+the+effects+of+superoxide+anion+inhibition+in+a+rat+sepsis+model.">Severity of sepsis alters the effects of superoxide anion inhibition in a rat sepsis model.</a> J. Appl. Physiol. 97 (4), 1349-1357 (2004).</li><li>Karzai, W., 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=Protection+with+antibody+to+tumor+necrosis+factor+differs+with+similarly+lethal+Escherichia+coli+versus+Staphylococcus+aureus+pneumonia+in+rats.">Protection with antibody to tumor necrosis factor differs with similarly lethal Escherichia coli versus Staphylococcus aureus pneumonia in rats.</a> Anesthesiology. 99 (1), 81-89 (2003).</li><li>Jespersen, B., Knupp, L., Northcott, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femoral+Arterial+and+Venous+Catheterization+for+Blood+Sampling,+Drug+Administration+and+Conscious+Blood+Pressure+and+Heart+Rate+Measurements.">Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements.</a> J. Vis. Exp. (59), e3496 (2012).</li></ol>

The authors have no potential conflicts of interest.

Investigating therapeutic approaches to a clinical model of illness necessitates the ability to accurately perform hemodynamic monitoring, thus requiring vascular access. In our model of sepsis, arterial catheterization provides systemic pressure monitoring and venous catheterization allows for the application of standard clinical therapy. Standard therapy for sepsis includes the infusion of antibiotics to treat bacterial infection and the infusion of fluids and vasopressors to maintain arterial pressure within the desired range. Both catheters are used for blood sampling. In this design, the application of programmable infusion pumps with automatic triggering of the vasopressor infusion rate based on real-time mean arterial pressures is an advance over the clinical setting which requires manual titration by the medical staff and in the pre-clinical setting makes the use of this treatment modality possible for studying large numbers of animals.
Some complications were experienced early in the development of the model. These included partial occlusion of both arterial and venous catheters from over tightening, ties on PU2 and PU3 components, (1-2%); misplacement of jugular venous catheter into a small branch, (&#60;1%); unexplained sudden death after recovery (no pulmonary thrombus or other organ damage evident), (1-2%) resolved by keeping isoflurane concentration 2-3.5% with O2 flow rate of 2 L/min. Other common surgical complications of hemorrhage, local infection and self-inflicted damage were not observed. Once surgical proficiency is attained, each procedure should be able to be completed in no more than 15 min. The rate of success measured as a healthy recovery and patent catheters, can be expected to exceed 90% once fully trained. An additional animal is included in each study to minimize the loss of data and the total number of animals needed and account for a failed catheter that may be &#8220;infusion only&#8221; or mortality due to complications.
The most critical step in this procedure is the design and placement of the catheters. The materials need to be soft at the tip so as not to pierce the vessel wall without crimping too easily. The body of the catheter has to be stiffer (than the tip) to be able to advance into the vessel sufficiently and be secured in place. Figure 1 shows the 3 diameters of tubing that are connected for the arterial catheter and 2 different diameter tubing connected for the venous catheters. These components are slid within each other to reduce catheter diameter at the tip and are glued together with anchors added to ensure placement of the catheter in the vessel or heart is maintained.
Once recovered and connected to the swivel and tether, the MAP and CVP waveforms should appear similar to the data represented in Figure 2. To maintain arterial catheter patency during monitoring, an hourly heparin saline flush (0.05 ml) should be performed. Continuous infusion maintains the venous catheter patency. During line connection and flush, close attention must be paid to insure there are no air bubbles in the line. An air bubble of &#8805;0.1ml can result in an embolism in the lung, brain or other organs. Hemodynamic waveforms are recorded and analyzed over the course of the study. When compressing time in the x axis, Figure 3 shows the changes in MAP and CVP over the course of 1 hr as well as the changes in rate and effect of vasopressor infusion.
The procedure has several potential limitations. In our studies, the catheters remain patent for up to 11 days. It is unknown how long the catheters would remain patent beyond this period. To fit the catheters as described, there is a minimum vascular size, which roughly correlates with rodent weight, requiring animals enrolled in this study to be greater than 200 g. The use of a smaller catheter diameter would result in increasing resistances to flow reducing patency.&#160;Attaining central venous pressure measures requires precise catheter placement in the atrium sufficient to account for size and growth during the study period and can be a challenge.
Once mastered, arterial and vascular catheterization can provide the basis for a wide variety of rodent models requiring hemodynamic monitoring, blood sampling, and infusion of fluids or therapies while minimizing any pain and distress during instrumentation or handling once recovered. In fact, in conjunction with a previous JOVE publication10 describing catheterization of the femoral vein, we have successfully performed this model with 3 implanted catheters.

Arterial and venous catheterization of rats has long been used in laboratory research.1, 2 Catheterization can be used for monitoring of hemodynamic parameters including systolic, diastolic and mean arterial pressure (MAP), heart rate (HR) and central venous pressure (CVP).3,4 In addition, these catheters allow the infusion of standard treatments or potential therapies as well as blood sampling to further analyze the underlying mechanism of an illness or treatment. Therefore, having vascular access in a small animal model is critical for studying clinical performance measures and treatment effects.
To study the underlying causes of critical illness, it is important to first develop a 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, a severe pulmonary infection which then develops into sepsis.3, 5 Monitoring hemodynamic measures and providing standard clinical treatment is critical to simulate routine supportive care. Standard clinical treatment includes infusion of antibiotics which aids in clearing the underlying infection and the administration of fluids and vasopressors, two therapies employed in septic patients to maintain blood pressure.5-7 Vascular access, specifically implantation of patent catheters is especially important when we study investigational therapies for sepsis.
For many years, the ability to administer standard clinical treatment to rodents was limited by the availability of the materials with the needed properties for constructing implantable catheters and the ability of the infusion technology to deliver small volumes accurately over time. In addition, the ability to trigger an infusion in response to the continuous monitoring of a specific hemodynamic measure allows for a consistent and reproducible standard treatment model when using larger sample sizes typical of rodent studies.8, 9 These technological limitations in materials and precision hardware have been overcome but to simultaneously provide routine therapies while monitoring hemodynamic parameters, both arterial and venous vascular access must be available.
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 used to connect the implanted catheters to the hemodynamic monitoring system. During the study, the system allows for a fluid infusion based on either delivering a given volume at a certain infusion rate or an automated system that delivers an infusion rate depending on the mean arterial pressure detected to maintain a given pressure range. The fluid infusion system uses programmable syringe pumps that interface with the data acquisition system&#8217;s digital outputs and are controlled by the software monitoring the MAP. The tether and swivel setup allows free movement of the rat during the study while monitoring pressures, infusing fluids and vasopressors, and performing blood sampling without having to manipulate the animals. Simultaneous monitoring of up to 12 instrumented animals with 2 catheters each (in our setup) with the hardware expandable to monitoring 24 instrumented animals allows for a great deal of flexibility for studying multiple stratified groups in each experiment.
This catheterization procedure can be beneficial to research facilities that use similar rodent models requiring vascular access for monitoring, sampling and treatment for up to 11 days. If the research facility considering this procedure has experience with rodents and a proper support structure for maintaining these animals then developing these skills can provide a significant cost saving, reducing total cost per catheterized rat from more than $180 to less than $50 (including na&#239;ve rat, catheter components and technical costs) and increasing scheduling flexibility (na&#239;ve rats are readily available vs. company delivery schedule of implanted rats). The success of this procedure is dependent on the skills of the individual performing the catheterizations. The training animals required to become proficient in this procedure has ranged from 20 to 40 rats with a resulting catheter patency rate of &#62;90%.

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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.

Changes in vascular pressure are transmitted through the fluid filled catheters (Figure 1) and converted into electrical signals represented by the hemodynamic waveforms (Figures 2, 3). Without vascular access, these measures could not be made. Real-time streaming of the waveforms allows for detection and analysis of changes on a beat to beat basis (Figure 2). Compressing the time scale of the waveforms allows for quantifying changes that occur over a longer duration (minutes to days) (Figure 3, upper 4 waveforms) which can be correlated with changes in vasopressor infusion rates (Figure 3, lower 4 waveforms).
<img alt="Figure 1" src="/files/ftp_upload/51881/51881fig1.jpg" /> 
Figure 1. Catheter Design: Details of the components of the arterial and venous catheters. PU, polyurethane; PE, polyethylene (segment length) <a href="https://www.jove.com/files/ftp_upload/51881/51881fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/51881/51881fig2.jpg" /> 
Figure 2. Real-time MAP waveforms of 4 typical rodents 7 days post-catheterization over 7 sec. <a href="https://www.jove.com/files/ftp_upload/51881/51881fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/51881/51881fig3.jpg" /> 
Figure 3. Time-compressed MAP waveforms of 4 typical rodents (upper 4 waveforms) 7 days post-catheterization over 1 hr. When the MAP is averaged (lower 4 waveforms), that value is used to trigger ADDS high (&#60; 90 mmHg), low (&#60;100 mmHg) or stop (&#62;110) flow response from the infusion pump. <a href="https://www.jove.com/files/ftp_upload/51881/51881fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model at JoVE.com

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

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. ...

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Research

JoVE Journal

Medicine

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).

Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically ...

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

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 ...

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.

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 ...

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 ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Microsurgical Skills of Establishing Permanent Jugular Vein Cannulation in Rats for Serial Blood Sampling of Orally Administered Drug

Blood sampling in small laboratory animals is necessary for pharmaceutical lead optimization but can cause great harm and stress to experimental animals, which could potentially affect results. The jugular vein cannulation (JVC) in rats is a widely used model for repeated blood collection but requires adequate training of surgery skills and animal care. This article details the microsurgical procedures for establishing and maintaining a permanent JVC rat model with specific focus on the placement and sealing of the jugular cannula. The importance of monitoring physiological (e.g., body weight, food, and water intake) and hematological parameters, was highlighted with results presented for 6 days post-surgery during the rat&#39;s recovery. The drug-plasma concentration-time profile of orally administered natural phenol ellagic acid was determined in the JVC rat model.

Detailed microsurgical techniques are demonstrated to establish a longer-term jugular vein cannulation rat model for sequential blood collection in the same animal. Physiological and hematological parameters have been monitored during the rat's recovery phase. This model has been applied to study pharmacokinetics of orally administered polyphenol without inducing animal stress.

Blood sampling in small laboratory animals is necessary for pharmaceutical lead optimization but can cause great harm and stress to experimental animals, ...