This work is supported by National Natural Science Foundation of China (No. 82003692) to R.X. Zhang; Top Academic Scholarship at Northwestern Polytechnical University to R. Miao.

The procedures described below were performed as part of a protocol approved by the Institutional Animal Care and Use Committee of Northwestern Polytechnical University (No. 202101117).
1. Preoperative preparation (the day before the surgery)
NOTE: Required solutions: normal saline (0.9% w/v sodium chloride), heparinized saline (1% w/v heparin sodium), catheter lock solution, non-steroidal anti-inflammatory drug (NSAID), such as meloxicam solution (2 mg/mL).
<ol>
	<li>Solution preparation
	<ol>
		<li>Aliquot 200 &#181;L of pre-packaged catheter lock solution in a 1.5 mL sterile microcentrifuge tube. 
		NOTE: Catheter lock solution is composed of heparinized saline (0.4% v/v heparin sodium) mixed with glycerol (v/v,1:1).</li>
		<li>Mix 1 g of heparin sodium in 100 mL of the normal saline to prepare 1% heparinized saline.</li>
		<li>Dissolve meloxicam in normal saline to prepare a 2 mg/mL concentration solution for postoperative pain relief. 
		NOTE: Prepared heparinized saline and meloxicam solution are filtered through a 0.22 &#181;m filter. All the solutions are sterilized and stored at 4&#176;C for future use.</li>
	</ol>
	</li>
	<li>Surgical instruments and materials
	<ol>
		<li>Pack all clean surgical tools in a pouch and tape it with a piece of autoclave sterilization tape. Refer to Figure 1A for the specific surgical instruments used.</li>
		<li>Autoclave the surgical pouch at 121 &#176;C for 30 min for the next day use.</li>
	</ol>
	</li>
	<li>Animal preparation
	<ol>
		<li>Prior to the surgery, house all male Sprague-Dawley (SD) rats in the standard Animal Room with controlled temperature at 22 &#177; 1 &#176;C. Feed them with the standard laboratory food and water ad libitum for at least 7 days. 
		NOTE: Both male and female rats can be used for the JVC model, and their typical ages and body weights vary from 9-14 weeks and 294 &#177; 57 g, respectively.</li>
		<li>Anesthetize the rat with 3%-3.5% isoflurane mixed with oxygen in a pre-anesthesia chamber. Determine whether the rat becomes unconscious by the lack of response to foot pinch.</li>
		<li>Gently take the rat out, place the rat&#39;s nose into an anesthetic nosepiece supplying 2%-2.5% isoflurane.</li>
		<li>In the ventral and dorsal position, remove&#160;the fur thoroughly around its right shoulder and posterior areas of neck with depilatory cream and a pet razor. Return the rat to the cage for surgery to be performed on the next day.</li>
	</ol>
	</li>
</ol>
2. Before the surgery on the day
<ol>
	<li>Prepare the aseptic workstation
	<ol>
		<li>Spray 75% medical alcohol to disinfect the operation area, and then place the heating pad covered with a clean cushion. Set the LED lamp with a cold light source beside the workstation.</li>
		<li>Pre-warm the required solutions (step 1.1) to room temperature.</li>
		<li>Fill 0.6 mL of heparinized saline and 0.15 mL of catheter lock solution in two sterile 1.0 mL blunt tipped syringes, respectively. Withdraw 2.5 mL of the normal saline using a sterile 5.0 mL syringe.</li>
		<li>Soak the cotton balls in 75% medical alcohol. Squeeze out excess ethanol before use.</li>
		<li>Weigh and record the rat&#39;s body weight.</li>
	</ol>
	</li>
</ol>
3. During the surgery
<ol>
	<li>Surgical preparation
	<ol>
		<li>Wear the surgical coat, sterile gloves, and facial mask. Then open the sterilized surgical pouch, leave all surgical tools in 75% medical alcohol, and dry them before use.</li>
	</ol>
	</li>
	<li>Jugular vein isolation 
	&#8203;NOTE: The estimated operation time for this part is 10 min.
	<ol>
		<li>Anesthetize the surgery-ready and shaved rat with 3%-3.5% isoflurane mixed with oxygen in an induction chamber and determine whether the rat becomes unconscious by the lack of response to foot pinch.</li>
		<li>Place the rat&#39;s nose into the nosepiece supplied with 2%-2.5% isoflurane to maintain the anesthesia.</li>
		<li>Subcutaneously inject (s.q.) meloxicam solution at a dose of 2 mg/kg.&#160; 
		NOTE: Make sure to select analgesics that do&#160;not&#160;interact&#160;with the drug compound of interest in the pharmacokinetics&#160;study.</li>
		<li>Using adhesive tape, restrain the rat&#39;s forearms in their ventral position to each side of the surgical platform.</li>
		<li>Gently scrub the surgical area by alternating between cotton balls soaked in 75% medical alcohol and iodine-based scrub for a total of three times.</li>
		<li>Carefully lift the skin near the clavicle on the right side of the midline of the neck with forceps and make an incision towards the chest about 1.5-2.0 cm in length with a pair of surgical scissors.</li>
		<li>Blunt dissect the thin tissue cover with iris scissors to expose the underneath jugular vein. The proximal cephalic end of the external jugular vein consists of two branches, which can be visually identified. 
		NOTE: Depending on the age and sex of the rat, the soft tissue (e.g., salivary glands, lymphatic nodes, and fatty tissues) covering the jugular vein varies. Compared to the young rats, the old rats are fatter (e.g., BW &#62; 300 g), and thus need more tissue separation before the jugular vein is visible.</li>
		<li>Lift the jugular vein along with its connective membranous tissues to visualize the lymph gland attached to the jugular vein. Carefully separate the vein along the vascular direction from surrounding muscle, fat, and other tissues.</li>
		<li>Nudge the forceps under the jugular vein without damaging the collateral blood vessels and pass two pieces of 6-0 suture under the vein to mark the two ends of the blood vessel individually.</li>
		<li>Pull one piece of the suture as far as possible toward the rat head and ligate the vein cranially with 2-3 knots using forceps.</li>
		<li>Place the second ligature on the caudal end of the vein with 1 loose knot.</li>
	</ol>
	</li>
	<li>Jugular vein cannulation 
	NOTE: The estimated operation time for this part is 15 min.
	<ol>
		<li>Open the package containing 11 cm polyurethane (PU) catheter (I.D. 0.6 mm x O.D. 0.9 mm, Figure 1B) and attach the catheter to the prepared blunt tipped syringe filled with the heparinized saline.</li>
		<li>Slowly push the heparinized saline into the catheter to avoid air bubbles.</li>
		<li>Nudge the non-tip flat side of the forceps under the jugular vein to exit on the other side. Make a small v-shaped cut on the vein near the cranial tie with a pair of castroviejo micro scissors and gently open the incision with the tip of the elbow vessel dilator forceps. 
		NOTE: Rinse the incision with pre-warmed normal saline (37 &#176;C) if a small amount of blood gushes out.</li>
		<li>Cut out the oblique opening of the front end of the jugular vein catheter. Clamp the oblique end of the tube with forceps and slide it into the jugular vein. 
		NOTE: This step may need another person to facilitate the catheter sliding.</li>
		<li>While advancing the catheter, slowly withdraw the elbow microsurgical forceps and clamp the outer surface of the vessel with forceps. 
		NOTE: If the right blood vessel is selected and the tip of the catheter is successfully slid into the blood vessel, the entire catheter insertion process should not feel any flow resistance.</li>
		<li>Stop inserting the catheter upon hitting the first blue mark of the PU tube (Figure 1B), which is approximately 3.0 cm in length.</li>
		<li>Secure the inserted catheter to the vein with both caudal and rostral ligatures using forceps.</li>
		<li>Thread a 6-0 suture through the exposed tissue on the right side of the incision using a suture needle (1/2 curved cutting, 12 mm) and tie the ligature with a hemostat.</li>
		<li>Bend the catheter at the second blue mark (Figure 1B) to bind with the same ligature (in step 3.3.8) and to avoid occluding the PU-tubing.</li>
		<li>Snip all the extra suture thread and close the catheter by replacing the blunt tipped syringe with a 22 G stainless-steel plug.</li>
	</ol>
	</li>
	<li>Catheter exteriorization 
	NOTE: The estimated operation time for this part is 10 min.
	<ol>
		<li>Place the rat in the dorsal position and gently clean the area between the scapulae with the cotton ball soaked in 75% medical alcohol.</li>
		<li>Make a very small incision at the center of the dorsal neck with surgical scissors. Through the dorsal incision, guide and gently push the trochar underneath the skin toward the ventral incision on the right side of the neck.</li>
		<li>Put the venous catheter into the trochar and then pull out and guide the venous catheter toward the dorsal incision.</li>
		<li>Secure the exteriorized catheter into the muscle layer in the same way with the suture (see the procedure in steps 3.3.8 and 3.3.9).</li>
		<li>Close the skin layer of ventral and dorsal incisions with the 6-0 nylon suture and suture needle (3/8 curved cutting, 17 mm). Swab all surgical incisions with iodophor. 
		NOTE: The wound-clips is an alternative method to close the skin incision.</li>
		<li>Remove the catheter plug by clasping the catheter with fingertips. Place a new blunt tipped syringe and slowly draw back the syringe to test the blood flow. 
		NOTE: Since the rat is in the supine position, one may not be able to obtain blood samples. Blood samples could be obtained by changing to a side body position.</li>
		<li>Hold the catheter again with fingertips and inject 0.2 mL of heparinized saline and 0.1 mL of lock solution into the catheter using the blunt tipped syringe.</li>
		<li>Hold the catheter with fingertips and replace the syringe with a stainless-steel plug. Unclamp the catheter and push the plug slightly in to ensure the tightness of the catheter.</li>
	</ol>
	</li>
</ol>
4. Immediate post-surgical care
<ol>
	<li>Recover the rat in the dorsal decubitus position by caging it&#160;individually with fresh corncob bedding. Often, provide a temperature-regulated heating pad to maintain the core body temperature. 
	NOTE: For animal welfare, leaving food and water on the bedding is an effective way to alleviate the pain caused by neck movements when eating and drinking.</li>
	<li>Record the end time of the surgery and monitor the rat at 2 h intervals for at least 4 h. Provide additional analgesia for the recovery if the rat shows signs of pain or distress.</li>
</ol>
5. Physiological and hematological monitoring during recovery phase
<ol>
	<li>Monitor the body weight and the food and water intake daily and record the data.&#160;</li>
	<li>To collect a small volume of fresh blood for hematological test, place the rat in a restrainer. Open the plug and insert the syringe into the venous PU catheter to ensure the catheter is not obstructed. 
	NOTE: The blood collection was performed at the same time daily for 6 consecutive days.</li>
	<li>Discard the initial withdrawn blood, which contains a mixture of blood, heparinized saline, and catheter lock solution.</li>
	<li>Use a new syringe to collect 150 &#181;L of fresh blood sample and transfer the blood sample to the 0.5 mL tube containing K2EDTA (1.8 mg/mL blood) spray dried on the tube wall. 
	NOTE: If the catheter is blocked, 0.2 mL of heparinized saline can be injected into the catheter to flush the catheter a few minutes before the next blood collection time.</li>
	<li>Inject sterile saline in the same volume to compensate for the withdrawn blood. Inject 150 &#181;L of pre-warmed normal saline (37 &#176;C) and infuse 0.2 mL sterile heparinized normal saline through the catheter.</li>
	<li>Inject 100 &#181;L of the lock solution into the catheter to ensure the sealing and sterility of the catheter before the next sample collection.</li>
	<li>Analyze the blood samples within 2 h of collection using an automated blood cell counter.</li>
</ol>
6. Repeated blood sampling for pharmacokinetic studies of oral administered drug
NOTE: Rats with weight gain &#62;10 g and stable hematological level are suggested to be enrolled for future study. Following the current protocol, the JVC rats required 4 to 6 days to recover.
<ol>
	<li>After 4-6 days of surgery, fast the rat for 12 h with free access to water. 
	NOTE: Depending on the experimental goal, fasting the animal is optional.</li>
	<li>Orally gavage the fasted rat with natural phenol bioactive ellagic acid at a dose of 6 mg/kg with a straight gavage needle16.</li>
	<li>Collect 200 &#181;L of blood samples in the heparinized tubes via the jugular vein cannula at pre-determined time points over 24 h post-oral administration. The blood collection process follows the procedure in step 5.5. 
	NOTE: The catheter does not need to be closed with the lock solution until the blood collection is completed.</li>
	<li>Immediately centrifuge the blood sample at 3000 x g at 4 &#176;C for 10 min.</li>
	<li>Analyze the extracted plasma sample by liquid chromatography-mass spectroscopy17,18.</li>
</ol>

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The authors have nothing to disclose.

Mastering the technique of vessel cannulation requires significant practice and learning the lesson from each operation. Christakis et al. using cumulative sum (CUSUM) analysis, found that a researcher needs to practice 200 rats over a period of one year before being ready for the PK evaluation of drug candidates20. Yet, the operating time required for the vein cannulation can be significantly reduced by the number of rats performed13,20. ...

Repeated acquisition of blood samples from small laboratory animals, such as rodents, guinea pigs, and rabbits, is an important aspect for pharmaceutical lead optimization and also for reducing the number of animals used in research1,2. The pipeline for developing new diagnostic tools and drug formulation (e.g., vaccine) requires access to different volumes of blood in order to evaluate their robustness and performance in vivo, such as pharmacokinetics (PK), toxicity, and sensitivity3,4,5.
The laboratory approach to blood sample collection is broadly classified into two types, surgical and nonsurgical6. The nonsurgical approach is relatively easy to grasp for the researcher, which includes common techniques, such as cardiac puncture, orbital sinus puncture, and bleeding of the saphenous and tail vein. Multiple blood sampling is possible by some non-surgical methods, but the sample volume is small and can cause physical wound and psychological stress to the animals1. On the other hand, the surgical approach is a favorite alternative to repeated venipuncture, and it involves placement of a temporary or permanent cannula in the blood vessels of animals7,8,9. The large blood volume could be repeatedly withdrawn through the cannula in conscious rats while avoiding the stress and pain due to the handling technique, restrain, and anesthesia7,8,10,11. However, the cannula implantation requires an experienced researcher with adequate training in order to successfully collect the blood.
Blood collection through jugular vein cannulation (JVC) in rats is the most widely used method to study the drug PK6,10,12,13. Yet, establishment of the JVC rat model needs careful practice of microsurgical skills and knowledge of postsurgical care and maintenance. Especially, after the surgery, the rat requires administration of analgesics and sufficient recovery time to reach stable physiological condition for further experiments13,14,15. Although the body weight gain (i.e., &#62;10 g) is a valid and commonly applied indicator for the rat&#39;s recovery, it is not uncommon that the rats have unexpected death postoperatively due to dehydration, infection, and inflammation, which could be subtle to notice at the early onset14,15. In addition, catheter obstruction in the JVC model remains to be an issue during the blood collection.
The present protocol has demonstrated in detail the microsurgical procedures for JVC in an anesthetized rat with specific focus on the identification, isolation, and cannulation of the jugular vein. The importance of physiological and hematological monitoring of the rats during the recovery phase is highlighted. Finally, serial blood samples were collected through the venous catheter to study the PK of the orally administered natural phenol ellagic acid with poor bioavailability (i.e., low systemic concentration) to verify the JVC rat model.

<table><tbody><tr><td>0.5 mL test tube containing EDTA anticoagulant</td><td>Xinkang</td><td>N/A</td><td>collecting blood samples for hematology test</td></tr><tr><td>0.5*20 mm 1.0-mL syringe</td><td>KLMEDICAL</td><td>N/A</td><td>washing or replacing the fluid with saline</td></tr><tr><td>0.6*28.5 mm 5.0-mL syringe</td><td>HD</td><td>N/A</td><td>Subcutaneous injection</td></tr><tr><td>1.0-mL Blunt tipped syringe (22G)</td><td>skillsmodel</td><td>S4-PKT22G</td><td>Inject the saline and collect blood samples through catheter</td></tr><tr><td>1.5 mL sterile microcentrifuge tube</td><td>Axygen</td><td>MCT-150-C-S</td><td>Store sterile catheter lock solution heparinized saline and meloxicam solution</td></tr><tr><td>1.5&amp;nbsp;mL microcentrifuge tubes</td><td>Biosharp</td><td>BS-15-M</td><td>blood collection</td></tr><tr><td>1/2&amp;nbsp; circle cutting 5*12 mm suture needle</td><td>skillsmodel</td><td>S4-FHZ</td><td>Thread the muscle layer to fix the catheter</td></tr><tr><td>3/8 circle cutting 7*17 mm suture needle</td><td>skillsmodel</td><td>S5-FHZ</td><td>Suture the incision of rat cortex</td></tr><tr><td>6-0 sterile non-absorbable silk suture thread</td><td>JUNSHENG</td><td>N/A</td><td>ligature</td></tr><tr><td>75% medical alcohol</td><td>HONGSONG</td><td>N/A</td><td>Disinfection</td></tr><tr><td>Adhensive tape</td><td>LIUTAI</td><td>N/A</td><td>positioning the rat</td></tr><tr><td>Autoclave sterilization tape</td><td>Biosharp</td><td>BS-QT-028</td><td>Mark sterilized items</td></tr><tr><td>Automated blood cell counter</td><td>Sysmex</td><td>XN-550</td><td>Hematology test</td></tr><tr><td>Castroviejo micro scissors</td><td>skillsmodel</td><td>WA1010</td><td>Cut the opening in the blood vessel</td></tr><tr><td>Centrifuge</td><td>Thermo Fisher Scientific</td><td>75002402</td><td>Plasma preparation</td></tr><tr><td>Clean cushion</td><td>Qingjie</td><td>N/A</td><td>Prepare the operation area</td></tr><tr><td>Cotton balls</td><td>HC</td><td>N/A</td><td>Wound disinfection and sterilization</td></tr><tr><td>Cotton swabs</td><td>BEITAGOGO</td><td>N/A</td><td>Disinfection</td></tr><tr><td>Curved hemostat</td><td>skillsmodel</td><td>N/A</td><td>ligature</td></tr><tr><td>DN50 Stainless-steel rat restrainer</td><td>skillsmodel</td><td>S4-RGDQ1</td><td>Restrict the movement of rats for easy operation</td></tr><tr><td>Ellagic acid</td><td>Aladdin</td><td>E102710-25g</td><td>natural phenol for oral administration</td></tr><tr><td>Half-curved forceps</td><td>skillsmodel</td><td>53072</td><td>Lift the muscle layer and tissue, isolate the jugular vein and tie the suture</td></tr><tr><td>Heating pad</td><td>Warm mate</td><td>N/A</td><td>preventing heat loss of animal</td></tr><tr><td>Heparin sodium</td><td>Solarbio</td><td>H8060</td><td>anticoagulant</td></tr><tr><td>Iodophor</td><td>Xidebao</td><td>N/A</td><td>Clean the wound</td></tr><tr><td>Iris scissors</td><td>skillsmodel</td><td>54002</td><td>Bluent separation the muscle layer</td></tr><tr><td>Isoflurane</td><td>RWD</td><td>R510-22-16</td><td>anaesthesia</td></tr><tr><td>LED lamp</td><td>EMPERORFEEL</td><td>N/A</td><td>sugery</td></tr><tr><td>Liquid chromatography-mass spectroscopy</td><td>Thermo Fisher Scientific</td><td>VQF01-20001/ TSQ02-10002</td><td>detection of drug concentration in plasma</td></tr><tr><td>Meloxicam</td><td>Hongqiang</td><td>N/A</td><td>Analgesic</td></tr><tr><td>Normal saline</td><td>KL</td><td>N/A</td><td>Prepara the solution and protect blood vessels from drying out</td></tr><tr><td>Pet razor</td><td>Codos</td><td>3180</td><td>Shaving the fur</td></tr><tr><td>Phosphate-buffered saline</td><td>ZHHC</td><td>PW012</td><td>Preparation of Ellagic acid solution</td></tr><tr><td>PU catheter</td><td>skillsmodel</td><td>RJVC-PU</td><td>Jugular vein cannulation</td></tr><tr><td>Small animal operation anesthesia console</td><td>RWD</td><td>68620</td><td>Operation workstation</td></tr><tr><td>Spray bottle</td><td>Other</td><td>N/A</td><td>aseptic workstation</td></tr><tr><td>Stainless steel plug (22G)</td><td>skillsmodel</td><td>S4-PKD22G</td><td>Plug the catheter to ensure its sealing</td></tr><tr><td>Stainless steel trochar</td><td>skillsmodel</td><td>S$-PKDGZ</td><td>Guide the catheter exteriorization</td></tr><tr><td>Sterile lock solution</td><td>skillsmodel</td><td>SK-FB</td><td>lock the catheter to ensure its sterility</td></tr><tr><td>Straight feeding needle</td><td>skillsmodel</td><td>N/A</td><td>Oral gavage</td></tr><tr><td>Surgical pouch</td><td>BKMAM</td><td>N/A</td><td>container for sterilization of surgical instruments</td></tr><tr><td>Surgical scissors</td><td>skillsmodel</td><td>J21070</td><td>Cut incision on rat skin</td></tr><tr><td>Vessel dilator balanced forceps</td><td>skillsmodel</td><td>WA3020</td><td>Expand the blood vessel and guide the cannula to slide in</td></tr><tr><td>ZS-MV Small animal anesthesia machine</td><td>ZSLab</td><td>1057003</td><td>inducing and maintaining anaesthesia</td></tr></tbody></table>

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.

This protocol has thoroughly demonstrated how to establish a long-term JVC model using microsurgical skills for serial blood collection. Figure 1A shows the essential surgical instruments and materials used to conduct the surgery. The specification of PU catheter with three blue marks is also illustrated, which is helpful for guiding the researcher to place the vein cannula in step 3.3., how to use the marks on the PU catheter to guide the cannulation (Figure 1B...

Watch this Scientific Journal Video about Microsurgical Skills of Establishing Permanent Jugular Vein Cannulation in Rats for Serial Blood Sampling of Orally Administered Drug at JoVE.com

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

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, ...

microsurgical-skills-establishing-permanent-jugular-vein-cannulation

Research

JoVE Journal

Medicine

7.9K Views.  Northwestern Polytechnical University. 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.

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

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available 1-3.
Here, we present two techniques for drawing blood from rats, each with its specific applications.
Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies 4,5, determining plasma chemistry, or blood counts 6.
Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro 4-9.

Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture.

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: ...

Biology

A General Method for Evaluating Deep Brain Stimulation Effects on Intravenous Methamphetamine Self-Administration

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people. There is a need for novel, effective and practical treatment strategies that are validated in animal models. Neuromodulation, including deep brain stimulation (DBS) therapy, refers to the use of electricity to influence pathological neuronal activity and has shown promise for psychiatric disorders, including drug dependence. DBS in clinical practice involves the continuous delivery of stimulation into brain structures using an implantable pacemaker-like system that is programmed externally by a physician to alleviate symptoms. This treatment will be limited in methamphetamine users due to challenging psychosocial situations. Electrical treatments that can be delivered intermittently, non-invasively and remotely from the drug-use setting will be more realistic. This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence. Methamphetamine dependence is rapidly developed in rodents using an operant paradigm of intravenous (IV) self-administration that incorporates a period of extended access to drug and demonstrates both escalation of use and high motivation to obtain drug.

This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence.

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people.  There is a need ...

Behavior

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 ...

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

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

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

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

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...

Establishment of Deep Hypothermic Circulatory Arrest in Rats

Deep hypothermic circulatory arrest (DHCA) is routinely applied during surgeries for complex congenital heart disease and aortic arch disease. The present study aims to provide a method for establishing DHCA in rats. To evaluate the impact of the DHCA process on vital signs, a normal temperature cardiopulmonary bypass (CPB) rat model without circulatory arrest was used as a control. As expected, DHCA led to a significant decrease in body temperature and mean arterial blood pressure. The blood gas analysis indicated that DHCA increased lactic acid levels but did not influence the blood pH and the concentrations of hemoglobin, hematocrit, Na+, Cl&#8722;, K+, and glucose. Furthermore, compared with the normal temperature CPB rats, the results of the transmission electron microscopy showed a mild increase in hippocampal autophagosomes in the DHCA rats.

This protocol presents the establishment of deep hypothermic circulatory arrest in rats, which can be applied to investigate systemic inflammatory response syndrome, ischemia/reperfusion injury, oxidative stress, neuroinflammation, etc.

Deep hypothermic circulatory arrest (DHCA) is routinely applied during surgeries for complex congenital heart disease and aortic arch disease. The present ...

Immunology and Infection

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

Dynamic analysis of blood components is of great importance in understanding cardiovascular diseases and their related diseases, such as myocardial infarction, arrhythmia, atherosclerosis, cardiogenic pulmonary edema, pulmonary embolism, and cerebral embolism. At the same time, it is urgent to break through the continuous heart blood sampling technique in live rats to evaluate the effectiveness of distinctive ethnic medicine therapy. In this study, a blood microdialysis probe was implanted in the right jugular vein of rats in a precise and noninvasive surgical procedure. Cardiac blood samples were then collected at a rate of 2.87 nL/min to 2.98 mL/min by connecting to an online microdialysis sample collection system. Even more momentously, the acquired blood samples can temporarily be stored in microdialysis containers at 4 &#176;C. The microdialysis-based online continuous blood collection program from rat heart has greatly guaranteed the quality of blood samples, advancing and invigorating the scientific rationality of the research on systemic cardiovascular diseases and evaluating ethnomedicine therapy from the perspective of hematology.

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

The present protocol describes a simple and efficient method for the real-time and dynamic collection of rat heart blood using the microdialysis technique.

Dynamic analysis of blood components is of great importance in understanding cardiovascular diseases and their related diseases, such as myocardial ...

Comparative Study of Blood Collection Techniques in Rats

Modified Tail Vein and Penile Vein Puncture for Blood Sampling in the Rat Model

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood collection in rats: the lateral tail vein puncture and the dorsal penile vein puncture, which offer significant advantages over other previously described techniques. This study shows that these two procedures allow for fast sampling (under 10 min) and yield sufficient blood volumes for most assays (202 &#956;L &#177; 67.7 &#956;L). The dorsal penile vein puncture must be done under anesthesia, whereas the lateral tail vein puncture can be done on a conscious, restrained animal.
Alternating these two techniques, therefore, enables blood draw in any situation. While it is always recommended for an operator to be assisted during a procedure to ensure animal welfare, these techniques require only a single operator, unlike most blood sampling methods that require two. Moreover, whereas these previously described methods (e.g., jugular stick, subclavian vein blood draw) require extensive prior training to avoid harm to or death of the animal, tail vein and dorsal penile vein puncture are rarely fatal. For all these reasons, and according to the context (e.g., for studies including male rats, during the perioperative or immediate postoperative period, for animals with thin tail veins), both techniques can be used alternately to enable repeated blood draws.

Author Spotlight: Context Dependent Blood Collection Methods in Rats 

Here, we present a protocol to offer rapid, easy, and reliable blood collection alternatives for the rat model. We describe three different blood sampling methods according to the context: tail vein puncture under anesthesia or on a conscious animal, and dorsal penile vein puncture under anesthesia.

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood ...

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 ...

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

Summary

Explore More Videos

Chapters in this video

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

A General Method for Evaluating Deep Brain Stimulation Effects on Intravenous Methamphetamine Self-Administration

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

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

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Establishment of Deep Hypothermic Circulatory Arrest in Rats

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

Modified Tail Vein and Penile Vein Puncture for Blood Sampling in the Rat Model

Subclavian Vein Blood Sampling in Conscious Rats