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%.

<table><tbody><tr><td>Straight micro dissecting forceps</td><td>Roboz</td><td>RS-8102</td><td>surgical instrument</td></tr><tr><td>Delicate straight hemostatic forceps</td><td>Roboz</td><td>RS-7114</td><td>surgical instrument</td></tr><tr><td>Delicate curved hemostatic forceps</td><td>Miltex</td><td>&amp;nbsp;7-4</td><td>surgical instrument</td></tr><tr><td>Serrefine curved, very delicate micro clamp</td><td>Roboz</td><td>RS-5471</td><td>surgical instrument</td></tr><tr><td>Micro dissecting hook</td><td>Miltex</td><td>19-220</td><td>surgical instrument</td></tr><tr><td>Angled on edge micro dissecting spring scissor</td><td>Roboz</td><td>RS-5618</td><td>surgical instrument</td></tr><tr><td>Light operating scissor</td><td>Roboz</td><td>RS-6750</td><td>surgical instrument</td></tr><tr><td>Scalpel handle and blade (#10)</td><td>Cincinnati Surgical</td><td>RS-9843</td><td>surgical instrument</td></tr><tr><td>Pack of 4-0 silk suture with curved needle</td><td>Ethicon</td><td>FS-2</td><td>surgical instrument</td></tr><tr><td>Straight micro suturing needle holder</td><td>Roboz</td><td>RS-6410</td><td>surgical instrument</td></tr><tr><td>Wound clip</td><td>Stoelting</td><td>59027</td><td>surgical instrument</td></tr><tr><td>Sterile gauze 2&quot;x2&quot;&amp;nbsp;</td><td>Dynarex</td><td>3362</td><td>consumable</td></tr><tr><td>Gauze 4&quot;x4&quot;&amp;nbsp;</td><td>Covidien</td><td>2556</td><td>consumable</td></tr><tr><td>Anesthesia vaporizer</td><td>Surgivet</td><td>V703001</td><td>equipment</td></tr><tr><td>Lucite box</td><td>Custom</td><td></td><td>equipment</td></tr><tr><td>Isoflurane</td><td>Baxter</td><td></td><td>equipment</td></tr><tr><td>Downdraft Table</td><td>Airscience</td><td></td><td>equipment</td></tr><tr><td>Table top surgical platform</td><td>Custom</td><td></td><td>equipment</td></tr><tr><td>Arterial Catheter</td><td>Custom - Scientific Commodities Inc.</td><td></td><td>consumable</td></tr><tr><td>Venous Catheter</td><td>Custom - Scientific Commodities Inc.</td><td></td><td>consumable</td></tr><tr><td>Data acquisition system</td><td>ADInstruments</td><td>Powerlab 16/30</td><td>equipment</td></tr><tr><td>Data analysis software</td><td>ADInstruments</td><td>LabChart v7.3</td><td>equipment</td></tr><tr><td>Programmable infusion pumps</td><td>Harvard Apparatus</td><td>PHD Ultra</td><td>equipment</td></tr></tbody></table>

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. ...

catheterization-carotid-artery-jugular-vein-to-perform-hemodynamic

Research

JoVE Journal

Medicine

71.8K Views.  National Institutes of Health. 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.

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

Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet specific experimental requirements. It is further enhanced by the use of radioactive tracers that can determine tissue specific insulin-stimulated glucose uptake as well as whole body glucose turnover.

The hyperinsulinemic-euglycemic clamp is the "gold standard" for the assessment of insulin action. Insulin is infused at a constant rate stimulating glucose uptake. The amount of exogenous glucose infused to counter this drop is indicative of insulin sensitivity. Here the procedure is performed on a conscious, unrestrained rat.

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, ...

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 ...

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

A Rat Model of Ventricular Fibrillation and Resuscitation by Conventional Closed-chest Technique

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic components of cardiopulmonary resuscitation in humans is herein described. The model was developed in 1988 and has been used in approximately 70 peer-reviewed publications examining a myriad of resuscitation aspects including its physiology and pathophysiology, determinants of resuscitability, pharmacologic interventions, and even the effects of cell therapies. The model featured in this presentation includes: (1) vascular catheterization to measure aortic and right atrial pressures, to measure cardiac output by thermodilution, and to electrically induce ventricular fibrillation; and (2) tracheal intubation for positive pressure ventilation with oxygen enriched gas and assessment of the end-tidal CO2. A typical sequence of intervention entails: (1) electrical induction of ventricular fibrillation, (2) chest compression using a mechanical piston device concomitantly with positive pressure ventilation delivering oxygen-enriched gas, (3) electrical shocks to terminate ventricular fibrillation and reestablish cardiac activity, (4) assessment of post-resuscitation hemodynamic and metabolic function, and (5) assessment of survival and recovery of organ function. A robust inventory of measurements is available that includes &#8211; but is not limited to &#8211; hemodynamic, metabolic, and tissue measurements. The model has been highly effective in developing new resuscitation concepts and examining novel therapeutic interventions before their testing in larger and translationally more relevant animal models of cardiac arrest and resuscitation.

This article describes a rat model of electrically-induced ventricular fibrillation and resuscitation by chest compression, ventilation, and delivery of electrical shocks that simulates an episode of sudden cardiac arrest and conventional cardiopulmonary resuscitation. The model enables gathering insights on the pathophysiology of cardiac arrest and exploration of new resuscitation strategies.

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic ...

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle proliferation and obliteration of pulmonary arterioles. This in turn results in right ventricular (RV) failure, with significant morbidity and mortality. Rodent models of PAH, in the mouse and the rat, are important for understanding the pathophysiology underlying this rare disease. Notably, different models of PAH may be associated with different degrees of pulmonary hypertension, RV hypertrophy and RV failure. Therefore, a complete hemodynamic characterization of mice and rats with PAH is critical in determining the effects of drugs or genetic modifications on the disease. 
Here we demonstrate standard procedures for assessment of right ventricular function and hemodynamics in both rat and mouse PAH models. Echocardiography is useful in determining RV function in rats, although obtaining standard views of the right ventricle is challenging in the awake mouse. Access for right heart catheterization is obtained by the internal jugular vein in closed-chest mice and rats. Pressures can be measured using polyethylene tubing with a fluid pressure transducer or a miniature micromanometer pressure catheter. Pressure-volume loop analysis can be performed in the open chest. After obtaining hemodynamics, the rodent is euthanized. The heart can be dissected to separate the RV free wall from the left ventricle (LV) and septum, allowing an assessment of RV hypertrophy using the Fulton index (RV/(LV+S)). Then samples can be harvested from the heart, lungs and other tissues as needed.

Pulmonary arterial hypertension (PAH) is a disease of pulmonary arterioles that leads to their obliteration and the development of right ventricular failure. Rodent models of PAH are critical in understanding the pathophysiology of PAH. Here we demonstrate hemodynamic characterization, with right heart catheterization and echocardiography, in the mouse and rat.

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle ...

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal blood time-activity concentration also known as arterial input function (AIF) is a key point, especially for the characterization of animal disease models and the introduction of newly developed radiotracers. The knowledge of radiotracer availability in the blood helps to interpret PET/CT-derived data of tissue activity. For this purpose, online blood sampling during the PET/CT imaging is advisable to measure the AIF. In contrast to manual blood sampling and image-derived approaches, continuous online blood sampling has several advantages. Besides the minimized blood loss, there is an improved resolution and a superior accuracy for the blood activity measurement. However, the major drawback of online blood sampling is the costly and time-consuming preparation to catheterize the femoral vessels of the animal. Here, we describe an easy and complete workflow for catheterization and continuous blood sampling during small animal PET/CT imaging and compared it to manual blood sampling and an image-derived approach. Using this highly-standardized workflow, the determination of the fluorodeoxyglucose ([18F]FDG) AIF is demonstrated. Further, this procedure can be applied to any radiotracer in combination with different animal models to create fundamental knowledge of tracer kinetic and model characteristics. This allows a more precise evaluation of the behavior of pharmaceuticals, both for diagnostic and therapeutic approaches in the preclinical research of oncological, neurodegenerative and myocardial diseases.

Here a protocol for continuous blood sampling during PET/CT imaging of rats to measure the arterial input function (AIF) is described. The catheterization, the calibration and setup of the system and the data analysis of the blood radioactivity are demonstrated. The generated data provide input parameters for subsequent bio-kinetic modeling.

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal ...

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, ...

Subclavian Vein Puncture Method for Repeated Blood Collection in Conscious Rats

Subclavian Vein Blood Sampling in Conscious Rats

There are several established methods for obtaining repeated blood samples from rats, with the most commonly employed methods being lateral tail vein sampling without anesthesia and jugular vein sampling with anesthesia. However, most of these methods require assistance and anesthetic equipment and sometimes pose difficulties in terms of blood collection or the poor quality of blood samples. In addition, these methods of blood collection consume significant time and human resources when repeated blood sampling is required for a large number of rats. This study presents a technique for repetitive blood sampling in non-anesthetized rats by a single proficient individual. Highly satisfactory blood samples can be obtained by puncturing the subclavian vein. The method demonstrated an impressive overall success rate of 95%, with a median time of merely 2 min from rat restraint to the completion of blood collection. Furthermore, performing consecutive blood collections within the designated range does not inflict any harm on the rats. This method is worth promoting for blood collection, especially in large-scale pharmacokinetic studies.

Author Spotlight: Developing an Efficient Blood Collection Method in Non-Anesthetized Rats 

Here we present a combination of effective rat restriction and subclavian vein puncture methods that enable rapid, safe, and repeated blood collection in rats without anesthesia.

There are several established methods for obtaining repeated blood samples from rats, with the most commonly employed methods being lateral tail vein ...

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

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