Name: microsurgical skills of establishing permanent jugular vein cannulation in rats for serial blood sampling of orally administered drug
Uploaded: 2021-12-14T13:00:00
Description: 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

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 ...

<ol>
	<li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> Journal of Pharmacology and Pharmacotherapeutics. 1 (2), 87-93 (2010).</li><li>Sadler, A. M., Bailey, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+refined+technique+for+taking+repeated+blood+samples+from+juvenile+and+adult+mice.">Validation of a refined technique for taking repeated blood samples from juvenile and adult mice.</a> Laboratory Animals. 47 (4), 316-319 (2013).</li><li>Zhang, R. 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=Coordinating+biointeraction+and+bioreaction+of+a+nanocarrier+material+and+an+anticancer+drug+to+overcome+membrane+rigidity+and+target+mitochondria+in+multidrug-resistant+cancer+cells.">Coordinating biointeraction and bioreaction of a nanocarrier material and an anticancer drug to overcome membrane rigidity and target mitochondria in multidrug-resistant cancer cells.</a> Advanced Functional Materials. 27 (39), 12 (2017).</li><li>Zhang, R. 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=Polymer-lipid+hybrid+nanoparticles+synchronize+pharmacokinetics+of+co-encapsulated+doxorubicin-mitomycin+C+and+enable+their+spatiotemporal+co-delivery+and+local+bioavailability+in+breast+tumor.">Polymer-lipid hybrid nanoparticles synchronize pharmacokinetics of co-encapsulated doxorubicin-mitomycin C and enable their spatiotemporal co-delivery and local bioavailability in breast tumor.</a> Nanomedicine-Nanotechnology Biology and Medicine. 12 (5), 1279-1290 (2016).</li><li>Zhang, R. 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=Sample+extraction+and+simultaneous+chromatographic+quantitation+of+doxorubicin+and+mitomycin+C+following+drug+combination+delivery+in+nanoparticles+to+tumor-bearing+mice.">Sample extraction and simultaneous chromatographic quantitation of doxorubicin and mitomycin C following drug combination delivery in nanoparticles to tumor-bearing mice.</a> Journal of Visualized Experiments: JoVE. (128), e11 (2017).</li><li>Bakar, S. K., Niazi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+reliable+method+for+chronic+cannulation+of+the+jugular+vein+for+pharmacokinetic+studies+in+rats.">Simple reliable method for chronic cannulation of the jugular vein for pharmacokinetic studies in rats.</a> Journal of Pharmaceutical Sciences. 72 (9), 1027-1029 (1983).</li><li>Harms, P. G., Ojeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+simple+procedure+for+chronic+cannulation+of+the+rat+jugular+vein.">A rapid and simple procedure for chronic cannulation of the rat jugular vein.</a> Journal of Applied Physiology. 36 (3), 391-392 (1974).</li><li>Thrivikraman, K. V., Huot, R. L., Plotsky, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jugular+vein+catheterization+for+repeated+blood+sampling+in+the+unrestrained+conscious+rat.">Jugular vein catheterization for repeated blood sampling in the unrestrained conscious rat.</a> Brain Research Protocols. 10 (2), 84-94 (2002).</li><li>Weeks, J. R., Davis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+intravenous+cannulas+for+rats.">Chronic intravenous cannulas for rats.</a> Journal of Applied Physiology. 19 (3), 540-541 (1964).</li><li>Goldkuhl, R., 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=Plasma+concentrations+of+corticosterone+and+buprenorphine+in+rats+subjected+to+jugular+vein+catheterization.">Plasma concentrations of corticosterone and buprenorphine in rats subjected to jugular vein catheterization.</a> Laboratory Animals. 44 (4), 337-343 (2010).</li><li>Steffens, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+frequent+sampling+of+blood+and+continuous+infusion+of+fluids+in+the+rat+without+disturbing+the+animal.">A method for frequent sampling of blood and continuous infusion of fluids in the rat without disturbing the animal.</a> Physiology &amp; Behavior. 4 (5), 833-836 (1969).</li><li>Terao, N., Shen, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+serum+protein+binding+and+pharmacokinetics+of+l-propranolol+in+the+rat+elicited+by+the+presence+of+an+indwelling+venous+catheter.">Alterations in serum protein binding and pharmacokinetics of l-propranolol in the rat elicited by the presence of an indwelling venous catheter.</a> Journal of Pharmacology and Experimental Therapeutics. 227 (2), 369-375 (1983).</li><li>Feng, J., 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=Catheterization+of+the+carotid+artery+and+jugular+vein+to+perform+hemodynamic+measures,+infusions+and+blood+sampling+in+a+conscious+rat+model.">Catheterization of the carotid artery and jugular vein to perform hemodynamic measures, infusions and blood sampling in a conscious rat model.</a> Journal of Visualized Experiments: JoVE. (95), e51881 (2015).</li><li>Karim, N., Ali, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jugular+vein+cannulation+in+rats+-+A+mini+review.">Jugular vein cannulation in rats - A mini review.</a> Canadian Journal of Pure and Applied Sciences. 3, 929-935 (2009).</li><li>Ling, S., Jamali, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cannulation+surgery+and+restraint+stress+on+the+plasma+corticosterone+concentration+in+the+rat:+application+of+an+improved+corticosterone+HPLC+assay.">Effect of cannulation surgery and restraint stress on the plasma corticosterone concentration in the rat: application of an improved corticosterone HPLC assay.</a> Journal of Pharmacy &amp; Pharmaceutical Sciences. 6 (2), (2003).</li><li>Lei, F., 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=Pharmacokinetic+study+of+ellagic+acid+in+rat+after+oral+administration+of+pomegranate+leaf+extract.">Pharmacokinetic study of ellagic acid in rat after oral administration of pomegranate leaf extract.</a> Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences. 796 (1), 189-194 (2003).</li><li>Yan, L. L., 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=Method+development+and+validation+for+pharmacokinetic+and+tissue+distributions+of+ellagic+acid+using+Ultrahigh+Performance+Liquid+Chromatography-Tandem+Mass+Spectrometry+(UPLC-MS/MS).">Method development and validation for pharmacokinetic and tissue distributions of ellagic acid using Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS).</a> Molecules. 19 (11), 18923-18935 (2014).</li><li>Long, J. F., 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=Bioavailability+and+bioactivity+of+free+ellagic+acid+compared+to+pomegranate+juice.">Bioavailability and bioactivity of free ellagic acid compared to pomegranate juice.</a> Food &amp; Function. 10 (10), 6582-6588 (2019).</li><li>Zhang, Y., 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=PKSolver:+An+add-in+program+for+pharmacokinetic+and+pharmacodynamic+data+analysis+in+Microsoft+Excel.">PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel.</a> Computer Methods and Programs in Biomedicine. 99 (3), 306-314 (2010).</li><li>Christakis, I., 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=Learning+curve+of+vessel+cannulation+in+rats+using+cumulative+sum+analysis.">Learning curve of vessel cannulation in rats using cumulative sum analysis.</a> Journal of Surgical Research. 193 (1), 69-76 (2015).</li><li>Nm, S., Oduola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematological+profile+shows+that+Inbred+Sprague+Dawley+rats+have+exceptional+promise+for+use+in+biomedical+and+pharmacological+studies.">Haematological profile shows that Inbred Sprague Dawley rats have exceptional promise for use in biomedical and pharmacological studies.</a> Asian Journal of Biomedical and Pharmaceutical Sciences. 4 (37), 33-37 (2014).</li><li>Lillie, L. E., Temple, N. J., Florence, L. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+values+for+young+normal+Sprague-Dawley+rats:+weight+gain,+hematology+and+clinical+chemistry.">Reference values for young normal Sprague-Dawley rats: weight gain, hematology and clinical chemistry.</a> Human &amp; Experimental Toxicology. 15 (8), 612-616 (1996).</li><li>He, Q. L., 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=Sex-specific+reference+intervals+of+hematologic+and+biochemical+analytes+in+Sprague-Dawley+rats+using+the+nonparametric+rank+percentile+method.">Sex-specific reference intervals of hematologic and biochemical analytes in Sprague-Dawley rats using the nonparametric rank percentile method.</a> PLoS One. 12 (12), 18 (2017).</li><li>EPA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+and+Documentation+of+Biological+Values+for+Use+in+Risk+Assessment.">Recommendations for and Documentation of Biological Values for Use in Risk Assessment.</a> U.S. Environmental Protection Agency. , (1988).</li><li>Gaud, N., 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=Single+jugular+vein+cannulated+rats+may+not+be+suitable+for+intravenous+pharmacokinetic+screening+of+high+logP+compounds.">Single jugular vein cannulated rats may not be suitable for intravenous pharmacokinetic screening of high logP compounds.</a> European Journal of Pharmaceutical Sciences. 99, 272-278 (2017).</li><li>Turck, D., 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=Clinical+pharmacokinetics+of+meloxicam.">Clinical pharmacokinetics of meloxicam.</a> Arzneimittel-Forschung/Drug Research. 47 (3), 253-258 (1997).</li><li>Aghazadeh-Habashi, A., Jamali, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+of+meloxicam+administered+as+regular+and+fast+dissolving+formulations+to+the+rat:+Influence+of+gastrointestinal+dysfunction+on+the+relative+bioavailability+of+two+formulations.">Pharmacokinetics of meloxicam administered as regular and fast dissolving formulations to the rat: Influence of gastrointestinal dysfunction on the relative bioavailability of two formulations.</a> European Journal of Pharmaceutics and Biopharmaceutics. 70 (3), 889-894 (2008).</li><li>Ludwig, E., 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=Activation+of+human+cytochrome+P-450+3A4-catalyzed+meloxicam+5+'-methylhydroxylation+by+quinidine+and+hydroquinidine+in+vitro.">Activation of human cytochrome P-450 3A4-catalyzed meloxicam 5 '-methylhydroxylation by quinidine and hydroquinidine in vitro.</a> Journal of Pharmacology and Experimental Therapeutics. 290 (1), 1-8 (1999).</li><li>Zhang, R. 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=Nanoparticulate+drug+delivery+strategies+to+address+intestinal+cytochrome+P450+CYP3A4+metabolism+towards+personalized+medicine.">Nanoparticulate drug delivery strategies to address intestinal cytochrome P450 CYP3A4 metabolism towards personalized medicine.</a> Pharmaceutics. 13 (8), (2021).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;mL microcentrifuge tubes</td>
			<td>Biosharp</td>
			<td>BS-15-M</td>
			<td>blood collection</td>
		</tr>
		<tr>
			<td>1/2&#160; 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, ...

Repeated blood sampling from the small laboratory animal is an important aspect of pharmaceutical lead optimization. This protocol details the microsurgical skills to establish a permanent jugular vein cannulation rat model. This technique can efficiently establish a jugular vein cannulation rat model and repeatedly collect blood samples through the cannula in conscious rats while avoiding stress and pain.It's important to locate the jugular vein and its cannulation as the visual demonstration of anatomical complexity is not available for this model. To begin, prepare the aseptic workstation with 75%medical alcohol, then anesthetize the shaved rat with isoflurane mixed with oxygen. Subcutaneously inject meloxicam solution at a dose of two milligrams per kilogram, then carefully lift the skin near the clavicle on the right side of the midline of the neck with forceps, and using a pair of surgical scissors, make a 1.5 to 2 centimeter long incision towards the chest.For exposing the underneath jugular vein, use iris scissors to blunt dissect the thin tissue cover. The proximal cephalic end of the external jugular vein consists of two branches which can be visually identified. Lift the jugular vein along with its connective membraneous tissue to visualize the lymph gland attached to the jugular vein.Carefully separate the vein along the vascular direction from the surrounding muscle, fat, and other tissues. 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. Pull one piece of the suture as far as possible toward the rat head and ligate the vein cranially with two to three knots using forceps.Place the second ligature on the caudal end of the vein with one loose knot. Attach an 11 centimeter polyurethane, or PU, catheter to the prepared blunt tip syringe filled with the heparinized saline, and slowly push the heparinized saline into the catheter to avoid air bubbles. 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 microscissors, and gently opened the incision with the tip of the elbow vessel dilator forceps. Cut out the oblique opening of the front end of the jugular vein catheter. Plant the oblique end of the tube with forceps and slide it into the jugular vein.While advancing the catheter, slowly withdraw the elbow microsurgical forceps, and clamp the outer surface of the vessel with forceps. Stop inserting the catheter upon hitting the first blue mark of the PU tube, which is approximately three centimeters in length. Secure the inserted catheter to the vein with both caudal and rostral ligatures using forceps.Thread a 6-0 suture through the exposed tissue on the right side of the incision using a suture needle, and tie the ligature with a hemostat. Then bend the catheter at the second blue mark to bind with the same ligature and avoid occluding the PU tubing. After snipping all the extra suture thread, close the catheter by replacing the blunt tipped syringe with a 22 gauge stainless steel plug.Place the rat in the dorsal position and gently clean the area between the scapula with the cotton ball soaked in 75%medical alcohol. Using the surgical scissors, make a very small incision at the center of the dorsal neck. Through the dorsal incision, guide and gently push the trochar underneath the skin toward the ventral incision on the right side of the neck.Put the venous catheter into the trochar and then pull out and guide the venous catheter toward the dorsal incision. After securing the exteriorized catheter into the muscle layer, close the skin layer of the ventral and dorsal incisions with the 6-0 nylon suture and suture needle and swab all surgical incisions with iodophor. Next, remove the catheter plug by clasping the catheter with fingertips.Place a new blunt tip syringe and slowly draw back the syringe to test the blood flow. Hold the catheter again with fingertips. Inject 0.2 milliliters of heparinized saline and 0.1 milliliters of lock solution into the catheter using the blunt tip syringe, and then replace the syringe with a stainless steel plug.Unclamp the catheter and push the plug in slightly to ensure the tightness of the catheter. To collect blood samples for hematological tests, 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. Discard the initially withdrawn blood containing a mixture of blood, heparinized saline, and catheter lock solution.Using a new syringe, collect 150 microliters of fresh blood sample and transfer the blood sample to a 0.5 milliliter tube previously sprayed with K2EDTA. Inject 150 microliters of pre-warmed normal saline, and infuse 0.2 milliliters of sterile heparinized normal saline through the catheter. Inject 100 microliters of the lock solution into the catheter to ensure the sealing and sterility of the catheter before the next sample collection.In the current study, the physiological and hematological conditions over six days postoperatively were investigated. The majority of the rats recovered within four to six days post surgery as evidenced by body weight gain of more than 10 grams, regular food intake, selected blood components relating to infection, dehydration, and inflammation, including white blood cell, red blood cell, hemoglobin, and platelet count. Thus, the rats required a minimum of four to six days for recovery after the procedure.The large intake of water indicated dehydration on the first day post-operation. In addition, the pharmacokinetics of the natural polyphenol ellagic acid was studied in the established jugular vein cannulation rat model and the poor bioavailability of the ellagic acid was indicated by its low plasma concentration over 24 hours. Correct isolation of the jugular vein is the most important first step because the jugular vein embedded within other soft tissues may not be immediately visible to the researcher.Sequential blood sampling within the same animal can be performed in the established JVC rat model. It's a useful and robust tool to evaluate drug formulations performance in vivo.

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Research

JoVE Journal

Medicine

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

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

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

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

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

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

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

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

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

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

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

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

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. ...

Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the tail vein is thin so that it is sometimes hard to puncture. In addition, the tail vein has low blood flow and requires a long sampling time to get sufficient blood. The present report describes a simple blood sampling method, the vacuum blood collection method, which is usually used to obtain blood samples from patients, here used for non-anesthetized rats. The 22 G butterfly needle tip was inserted into one of the lateral tail veins approximately 2-3 cm from the tip of the tail at an angle of approximately 20&#176;, and blood was collected in the vacuum collection tube by inserting the rubber end of the butterfly needle into the vacuum blood collection tube. The present experimental results show that the success rate was 95% in the experimental group and 90% in the beginner group. The success rate and puncture times were similar between two groups. The sampling duration was significantly shorter in the experimental group compared to beginner group. In conclusion, this vacuum blood collection method for sequential blood sampling from the tail vein of non- anesthetized rats is feasible and easy-to-learn, which might serve as a reliable alternative to other conventionally used blood sampling techniques for rats.

Here, we describe a simple tail vein blood sampling method in non-anesthetized rats using a vacuum extraction tube system. This method reduces the risk of direct exposure to blood and simplifies taking multiple samples from a single venipuncture.

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the ...

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 ...

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 ...

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 ...

Internal Jugular Vein Identification and Catheter Exit Site Preparation

To begin, place the anesthetized swine on the operating table. In the ventral field, make a four-centimeter incision between the trachea and the medial border of the sternocleidomastoid muscle. Divide the platysma and dissect the connective tissue to expose the internal jugular vein, or IJV, on the lateral border of the sternocleidomastoid muscle.Then isolate three to four centimeters of the IJV by dividing its branches with 4.0 coated and braided non-absorbable suture ties. Circumferentially dissect away from the surrounding connective tissue. At the cranial end of the IJV, pass a coated and braided non-absorbable suture tie twice underneath the vessel to create a loop around it.At the coddle end, pass a suture tie once underneath the vessel to create a sling. For catheter exit site preparation. Reposition the swine via lateral tilt toward the non-surgical side to expose the ipsilateral dorsal surgical field.Once the limbs are resecured, with a number 10 blade scalpel, make a 0.5 centimeter puncture in the skin at the desired catheter exit site, three centimeters lateral to the vertebral column, and five centimeter coddle to the head.