A MODIFIED RAT MODEL FOR CANNULATION AND COLLECTION …

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Lymphology 44 (2011) 82-88

A MODIFIED RAT MODEL FOR CANNULATION AND COLLECTION OF THORACIC DUCT LYMPH

Y. Li, J.-G. Wang, Y.-P. Li, Z.-F. Lin

Departments of Emergency (YL,Z-FL) and Neurology (Y-PL), Changzheng Hospital, Second MilitaryMedical University, Shanghai and Department of Biochemistry (J-GW), School of Basic MedicalSciences, Wenzhou Medical College, Wenzhou, China

ABSTRACT

Difficulty in collecting lymph samples insmall animals has impeded studies onlymphatic function and lymph composition.Here we report a simple and effective modifiedrat model for thoracic duct lymph drainagewhere animals remain in full consciousnessand have free movement and access to waterand food over 12 hours. The operative proce-dure required approximately 30 minutes toperform. Mean lymph drainage was 0.71±0.33ml/h, and protein concentration did notchange significantly (mean 37±2.59mg/ml)over the 12 hours. However, the number oflymphocytes fluctuated widely between0.08±0.03x106/ml and 12.17±6.58x106/ml. This modified animal model of thoracic ductlymph collection avoids influences of lipidintake, general anesthesia, or limited activityof animals on experimental outcomes, andtherefore more accurately reflects lymph flowand composition under normal physiologicalconditions.

Keywords: thoracic duct cannulation, ratmodel, lymph, lymph flow

The lymphatic system plays importantroles in body fluid homeostasis, lipidabsorption, metastasis, and immune functionas well as a key component in maintainingnormal interstitial fluid volume and protein

concentration (1). Disturbances from severeinjury, burns, and peritonitis may causesystemic inflammatory response and multipleorgan dysfunction (2), and these are reflectedin lymph flow and composition as are avariety of other disorders.

Various animal models of lymph drainagesampling the gastrointestinal lymphatic routehave been used for evaluating drug absorptionand distribution. Many of these studies havebeen initiated in rats because experiments in large animals are now more restricted bybioethical and economic considerations.Advantages of these models include thatdrugs absorbed directly into the lymphaticsystem can avoid the first-pass effect throughthe liver, allowing absorption and distributionof lipid-soluble drugs to be studied moreeffectively. In addition, immunoregulatoryfactors or chemicals used for treatment ofmetastatic cancers could also achieve optimaltherapeutic effects if they are delivereddirectly through the lymphatic system andnot the blood vascular route.

In the present study, we modified aprevious lymph drainage method (3) for asimpler and more effective thoracic ductlymph collection in which the rats are fullyconscious and have free movement and accessto water and food providing a method forlong-term study of the lymphatic circulationwith minimal interferences. It is not nece-ssary to feed fat to the rats in advance,

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY.

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restrain them by means of general anesthesia,or use intravenous fluid replacement orgastric gavage. Thus, this model minimizesfactors that may influence formation andcomposition of thoracic duct lymph andensures examination, intervention, andtreatment of the animals in a situation closerto the physiological condition.

MATERIALS AND METHODS

Animals

Twenty clean adult male SD ratsweighing 180 -200g (Shanghai Animal Centerof the Chinese Academy of Sciences) wereacclimated in our laboratory for one weekand fed with normal diet in a 12-h dark/lightcycle. The protocol of the animal experimentwas approved by the Ethics Committee of the Second Military Medical University(Shanghai, China). All procedures werecarried out under aseptic conditions inaccordance with the “Principles of Laboratory

Animal Care” (NIH publication No. 85-23revised 1985).The protocols for anesthesia,postoperative care, and sacrifice wereidentical for all animals. Rats wereanesthetized by intraperitoneal injection of10% chloral hydrate at 0.35mL/100g bodyweight and sacrificed at 24 h by anintravenous overdose of potassium chloride.

Procedures

One end of a 10-cm PE tube (ID 0.58mm,OD 0.99mm) was immersed in hot water to form a “U” shape with an angle of 180°. The “U”-shaped end was approximately1.5cm long, and a beveled face (~30°) wasmade on the open end. The other end wassecurely connected to a disposable vacuumblood collection needle (Fig. 1). This preparedtube system was sterilized with 60% ethyleneoxide for use.

The skin around the surgical site wasprepared and sterilized with iodophor twotimes. A 4-cm median incision was made on

Fig. 1. The lymph collecting system consists of the PE collection tube with the beveled edge on the U curve (A) whichis securely connected to a blood collection needle (B). The needle is then inserted into a standard blood collectiontube (C).


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the upper 2/3 abdomen between the xiphoidprocess and the pubic symphysis to open theabdominal cavity and reach the retroperi-toneal space by separating the perirenalfascia from the left kidney laterally. The leftkidney and adrenal gland were isolatedmedially, and the soft tissue was separatedalong the surface of the psoas major muscle.The left kidney, adrenal gland, small intes-tine, and spleen were wrapped with warmsaline-soaked gauze to prevent dehydration.Fascia and small vessels running across thesurface of the abdominal aorta weredissected, ligated, and cut away from thesurface of the abdominal aorta. The cream-colored cisterna chyli and thoracic duct wereexposed by pushing the abdominal aortarightward. The abdominal aorta and thethoracic duct were separated bluntly, and thethoracic duct was separated from the muscle.Two 5-0 Vicryl silk sutures were passedthrough under the thoracic duct, with oneplaced near the crura of the diaphragm andthe other near the cisterna chyli (Fig. 2A).

Avoiding the major vessels and nerves, a20-gauge needle was passed through the leftabdominal wall to introduce the PE catheterinto the abdominal cavity. The catheter wassutured intermittently by 3-4 stitches with a5-0 silk thread and fixed on the psoas majormuscle to prevent displacement or dislodge-

ment during cannulation. The length andangle of the catheter were adjusted properlyto avoid excessive tension after placement ofthe catheter in the thoracic duct. The thoracicduct was pricked by the tip of a 1ml syringeneedle, and the beveled end of the cannulawas placed carefully through the puncturesite into the thoracic duct and then into thecisterna chyli. Following insertion, one of thesilk sutures was tied, and the vacuum bloodtube connected. When the cream-coloredlymph was seen flowing quickly into thecatheter, the second suture was tied toprevent lymph reflux (Fig. 2B).

After assuring no significant activebleeding, the abdominal cavity was irrigatedwith warm normal saline. The left renalfascia was sutured intermittently and fixed onthe retroperitoneal wall, and the peritoneumand the abdominal wall was closed intermit-tently with two layers of 5-0 silk thread. Therat was then placed in a prone position andrubber loops to hold the vacuum bloodcollection tube were made and sutured ontothe midline of the cervical back of the animal.The blood collection needle was sutured andfixed on the rat back, and the positionbetween the blood collection tube and theneedle adjusted properly (Fig. 3). Lymph wascollected for 12 hours with a change of thecollection tube at every 2 hour time point.

Fig. 2. Anatomy of abdominal thoracic duct and cisterna chyli in rat as seen from the left lateral approach. Beforecannulation (A), the cisterna chyli, thoracic duct, and aorta have been isolated and cleared of crossing vessels.Sutures have been placed below the thoracic duct in preparation for ligation. After cannulation (B), the collectiontube can be seen as it enters the thoracic duct and passes the ligature into the cisterna chyli. The suturing of thecollection tube to the muscle provides stability.


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Precautions during the procedure

Aseptic technique is required and allmaterials sterilized before use. Surgicalprocedures should be performed gently,maintaining distance from the cisterna chyliwhen separating the thoracic duct to preventpossible injury to its wall. If lymph leakageoccurs due to injury, it can be stopped byapplying pressure with a cotton swab forseveral minutes. There are usually smallvessels such as the lumbar artery runningacross the thoracic duct, which may interferewith the procedure. These small vessels canbe ligated and cut off to facilitate exposure of the thoracic duct.

After successful cannulation, lymph canbe seen quickly flowing into the catheter assoon as the collection tube is connected, andthe cisterna chyli empties quickly. If thecisterna chyli is dilated or there is slow flowof lymph into the catheter, this finding mayindicate that the catheter is placed improperly.In this case, it is necessary to adjust thecatheter until the cisterna chyli can be seen

to empty. The lymph collection system should be fixed properly to the rat. If it is not,it may become crimped and affect lymphdrainage. Long-term obstruction may causelymph coagulation and loss of flow.

RESULTS

The model has proven successful incollecting thoracic duct lymph over 12 hoursin awake animals. Nineteen of the 20 animalsin this study were cannulated and the flow was consistent over the 12 hours with an average collection of 0.71±0.33 ml/h(mean±SD) (Fig. 4). The protein concen-tration did not change significantly over the12 hours at 37±2.59mg/ml (mean±SD) (Fig. 5). However, the number of lymphocytesdid fluctuate significantly between0.08±0.03x106 and 12.17±6.58x106/ml overthe 12 hours of the experiment (Fig. 6).

DISCUSSION

There are some differences between the

Fig. 3. The exteriorized collection tube and insertion of collection needle into the attached collection tube is shownin this fully awake rat.


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Fig. 4. Mean±SD lymph collection (ml) rates at time points 2-12 hours.

Fig. 5. Mean±SD of protein concentration in lymph at time points 2-12 hours.

flow of lymph drainage found in our studyand other studies previously reported in theliterature (3,4). The reason may be that theSD rats used in our study were relativelysmall (180-220g), while other studies usually

used rats weighing 300-350g. Rat size doeshave a direct influence on the total produc-tion of lymph. Boyd et al (4) reported that theamount of lymph drainage from the thoracicduct was 12.5±2.5ml/h. The difference may


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relate to their use of olive oil 1 hour beforeoperation and the fluid replacement givenduring operation. Feeding fat in the initialstage of experiments helps to identify thelocation and anatomic structure of thethoracic duct, and increased lymph flow byfluid replacement creates a favorablecondition for placement of the thoracic duct.However, large amounts of fat intake mayinterfere with later research measurements onlymph, and the model may not truly reflectlymph under normal physiologic conditions.We feel it is preferable not to feed fat beforeoperation as long as the anatomic structure ofthe thoracic duct is clearly understood, andcannulation is skillfully applied. In addition,the amount of lymph drainage is closelyrelated to food/water intake. Recognizing thatour animals would resume consciousness inabout 1 hour after operation and that theirfree access to food and water would increaseor influence the flow, we did not give anyfluid replacement to the animals in our study.

Lymph protein concentration is lowerthan that in the blood. In our study, lymphprotein concentration remained relativelystable within the 12 hours of the experiment,

and it did not decrease with the increasedamount of lymph drainage. These datasupport the stability of the model for researchpurposes.

One might think that since the number of lymphocytes in the blood of normal rats isrelatively stable that the number of lympho-cytes in lymph should also be fairly stable. In our study, the number of lymphlymphocytes was slightly lower in the earlystage of lymph drainage with the lowest levelof 1.5±1.2x106/ml at the 4-hour point onaverage. The number of lymphocytes quicklyrose again to a mean of 12.2±6.6x106/ml at 6-8 hours possibly related to a feedbackresponse with decreased number of lympho-cytes in the circulating blood mobilizinglymphocytes from the thymus, spleen, andother central lymphatic organs to enter thelymphatic system and causing the number oflymphocytes in lymph to rise markedly. Aftera transient rise, the number of lymphocytesbegan decreasing sharply at 10 hours. Themechanism of this great fluctuation oflymphocytes in lymph is unclear and needsfurther investigation.

Fig. 6. Mean±SD number of lymphocytes (106/ml) in lymph at time points 2-12 hours.


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Preparation of the rat thoracic-ductcannulation model is relatively difficult dueto its deep anatomic position and limitedspace for operation. In our study, we preparedthe model by entering the retroperitonealspace lateral to the left kidney, whichfacilitates finding the thoracic duct on the left side of the abdominal aorta. According tothe literature, the thoracic duct has threedifferent locations: at the left posterior, rightposterior, or behind abdominal aorta (5), andthe latter two are anatomic variations. In ourstudy, the thoracic duct was located in leftand posterior to the abdominal aorta in 16rats; posterior in 3; and right and posterior in1. The length of the thoracic duct from thecisterna chyli to the diaphragm crura averaged0.8cm (0.6-1.0cm), and anatomic variationsrendered operation more difficult. The onefailed case was mainly due to the unexpectedstructure of the cisterna chyli and thinness ofthe thoracic duct. A second cannulation wasattempted but was not successful.

Anticoagulants were used beforeoperation in most previous similar studies. In our study, we did not give the animals anyanticoagulants, and only used ethylenediamine tetraacetic acid (EDTA) to treat thecollection tube since lymph in the collectiontube would coagulate without anticoagulationtreatment. Knowing that injection of heparinand other anticoagulants would to someextent cause intraoperative and postoperativehemorrhage and alter the normal physiologicstatus of the rats, we tried to avoid possibleinfluencing factors during collection oflymph. As observation of our model lasted foronly 12 hours, it is not known whetheranticoagulation is necessary for longer timeperiods of drainage.

The lymph collection system used in thepresent study is original in that it was donewithout general anesthesia or restraint of theanimals, which are stressful maneuvers andmay interfere with normal physiologicconditions. Our system makes use of negativepressure of the vacuum blood collection tubeto collect lymph in a closed circuit when the

animals are fully conscious and freelymovable with free access to water and food,thus minimizing possible interferences withthe physiologic activities of the animals. Theslight negative pressure in the collection tubecould be thought to increase flow, but ourflow rates were consistent over time andlower than others reported.

This thoracic duct lymph drainage modelcan be useful for the study of immunoregu-lation, drug absorption and distribution, andlipid transport and metabolism. As theintestinal tract plays an important role in the pathogenesis of sepsis, this modifiedmodel may also facilitate exploration of the pathogenesis of sepsis through the lymphaticsystem.

REFERENCES

1. Bridenbaugh, EA, AA Gashev, DC Zawieja:Lymphatic muscle: A review of contractilefunction. Lymphat. Res. Biol. 1 (2003), 147-158.

2. Gosain, A, RL Gamelli: Role of thegastrointestinal tract in burn sepsis. J. BurnCare Rehabil. 26 (2005), 85-91.

3. Ionac, M, T Laskay, D Labahn, et al:Improved technique for cannulation of themurine thoracic duct: A valuable tool for thedissection of immune responses. J. Immunol.Methods 202 (1997), 35-40.

4. Boyd, M, V Risovic, P Jull, et al: A stepwisesurgical procedure to investigate thelymphatic transport of lipid-based oral drugformulations: Cannulation of the mesentericand thoracic lymph ducts within the rat. J.Pharmacol. Toxicol. Methods 49 (2004), 115-120.

5. Ionac, M: One technique, two approaches, and results: thoracic duct cannulation insmall laboratory animals. Microsurgery 23(2003), 239-245.

Zhao-fen Lin, PhDProfessor, Emergency DepartmentChangzheng HospitalSecond Military Medical UniversityNo. 415 Fengyang RoadShanghai 200003, China Tel: +8613601605100E-mail: [email protected]


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A MODIFIED RAT MODEL FOR CANNULATION AND COLLECTION …