www.elsevier.com/locate/jpharmtox

Journal of Pharmacological and Toxicological Methods 49 (2004) 115–120

Original article

A stepwise surgical procedure to investigate the lymphatic transport of

lipid-based oral drug formulations: Cannulation of the mesenteric and

thoracic lymph ducts within the rat

Michael Boyda, Verica Risovicb, Philip Julla, Eugene Choob, Kishor M. Wasanb,*

aAcute Care Animal Unit, Koerner Pavilion, University of British Columbia, Vancouver, BC, CanadabDivision of Pharmaceutics and Biopharmaceutics, Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall Avenue,

Vancouver, BC, Canada V6T 1Z3

Received 8 September 2003; accepted 24 November 2003

Abstract

Introduction: A number of animal models have been described for the assessment of intestinal lymphatic drug transport. Lymphatic

transport studies are commonly first conducted in the laboratory rat, with larger more complicated models (i.e., dog or pig) subsequently

investigated. However, the utility of lymph fistulation in large animals is limited by considerable logistical and economic constraints.

Methods: This paper describes a stepwise surgical procedure for cannulating the thoracic and mesenteric lymph ducts in male Sprague–

Dawley rats. Results: Following surgery, thoracic and mesenteric lymph flow rates during the 24-h period immediately following surgery

averaged 12.5F 2.5 and 2.4F 1.1 ml/h, respectively. This flow rate is greater than that obtained with previously described methods, which

require restraint of the animals and/or a 24-h recovery period and are reported to produce average intestinal lymph flow rates of 2 ml/h.

Discussion: This animal model can be utilized for the assessment of drug transport by the lymphatics and for determining what percentage of

lymphatic transport is a result of only intestinal lymphatics.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Mesenteric lymph duct; Thoracic lymph duct; Rat; Surgical procedure; Lymphatic drug delivery

1. Introduction ing general systemic drug exposure (Porter & Charman,

The gastrointestinal lymphatic system is a specific trans-

port pathway through which dietary lipids (Borgstrom,

Dahlqvist, Lundh, & Sjoval, 1957; Shiau, 1981; Thomson,

Keelen, Garg, & Clandinin, 1989; Thomson, Schoeller,

Keelen, Smith, & Clandinin, 1993), fat-soluble vitamins,

and water-insoluble compounds (i.e., halofantrine, ontazo-

last, amphotericin B) can gain access to the systemic circu-

lation (Hauss et al., 1998; Holm, Porter, Mullertz,

Kristensen, & Charman, 2002; Porter & Charman, 2001;

Wasan, 2002). Drugs transported by way of the gastrointes-

tinal lymphatic system bypass the liver and avoid potential

hepatic first-pass metabolism. Lymphatic delivery of immu-

nomodulatory agents and low therapeutic index drugs used

in the treatment of cancer cell metastases and HIV present an

opportunity to maximize therapeutic benefit while minimiz-

1056-8719/$ – see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.vascn.2003.11.004

* Corresponding author. Tel.: +1-604-822-4889; fax: +1-604-822-

3035.

E-mail address: Kwasan@interchange.ubc.ca (K.M. Wasan).

2001; Tucker, 1993). Furthermore, lymphatic drug transport

may promote drug incorporation into the body’s lipid-han-

dling system, thus offering the potential to manipulate drug

distribution and residence time within the body.

The majority of orally administered drugs gain access to

the systemic circulation by direct absorption into the portal

blood (Porter & Charman, 2001;Wasan, 2002). However, for

some water-insoluble compounds, transport by way of the

intestinal lymphatic system may provide an additional route

of access to the systemic circulation (Porter & Charman,

2001; Wasan, 2002). Exogenous compounds absorbed

through the intestinal lymph appear to be generally trans-

ported in association with the lipid core of intestinal lip-

oproteins (predominantly triglyceride-rich chylomicrons),

thereby requiring coadministered lipid to stimulate lipopro-

tein formation. Delivery into the bloodstream by way of the

intestinal lymphatics has been suspected to contribute to the

overall absorption of a number of highly lipophilic com-

pounds (Adachi, Liu, Horkoshi, & Ueno, 1993; Barnwell et

al., 1992; Gallo-Torres, 1970; Hauss, Mehta, & Radebaugh,

M. Boyd et al. / Journal of Pharmacological and Toxicological Methods 49 (2004) 115–120116

1994; Horst et al., 1976; Sugihara & Furuuchi, 1988; Sugi-

hara, Furuuchi, Ando, Takashima, & Harigaya, 1988), in-

cluding cyclosporine (Ueda, Lemaire, Gsell, & Nussbaumer,

1983), naftifine (Grimus & Schuster, 1984), probucol (Palin

&Wilson, 1984), mepitiostane (Ichihashi, Kinoshita, Shima-

mura, & Yamada, 1991; Ichihashi, Kinoshita, Takagishi, &

Yamada, 1991; Ichihashi, Kinoshita, Takagishi, & Yamada,

1992a; Ichihashi, Kinoshita, Takagishi, & Yamada, 1992b;

Ichihashi, Kinoshita, & Yamada, 1991), halofantrine (Holm

et al., 2002; Humberstone, Porter, & Charman, 1996; Porter,

Charman, & Charman, 1996; Wasan et al., 1999), and poly-

chlorinated biphenyls (Busbee, Yoo, Norman, & Joe, 1985).

Lymph from the intestinal lymphatic system (as well as

hepatic and lumbar lymph) drains through the thoracic lymph

duct into the left internal jugular vein and then to the systemic

circulation (Davidson, 1994; Johnson, 1976; Tso, 1994).

Thus, the transport of drug by way of the intestinal lymphatic

system may increase the percentage of drug that can gain

access to the systemic circulation. In addition, the process of

intestinal lymphatic drug transport often continues over time

periods longer than typically observed for drug absorption

through the portal vein. Consequently, drug transport

through the lymph may be utilized to prolong the time course

of drug delivery to the systemic circulation.

A number of animal models have been described for the

assessment of intestinal lymphatic drug transport (Charman

& Porter, 1996; Porter & Charman, 1997; Porter & Charman,

2001; White, Story, & Barnwell, 1991). Lymphatic transport

studies are commonly first conducted in the laboratory rat,

with larger more complicated models (i.e., dog or pig)

subsequently investigated (Charman & Porter, 1996; Porter

& Charman, 1997; Porter & Charman, 2001; White et al.,

1991). However, the utility of lymph fistulation in large

animals is limited by considerable logistical and economic

constraints. Ideally, sampling strategies for lymphatic trans-

port studies should provide the capacity to estimate both the

extent of lymphatic transport, as well as the extent of portal

blood absorption in order to estimate the overall bioavail-

ability of the drug/formulation. This strategy enables the

unambiguous determination of the extent of lymphatic

transport, relative to absorption via the portal blood, and

the total bioavailability of the drug/formulation. As lymphat-

ic transport can be affected by experimental factors such as

the site of lymphatic cannulation the period of fasting prior to

dosing (Charman & Porter, 1996; Kararli, 1995; Levet-

Trafit, Gruyer, Marjanovic, & Chou, 1996; Liu, Adachi,

Horkoshi, & Ueno, 1995; Porter & Charman, 1997; Porter &

Charman, 2001; White et al., 1991), it is important to

standardize procedures when comparing between studies.

Previously reported methods for collecting lymph from

the rat required total restraint of the animal and fluid

replacement, by intravenous or intraduodenal infusion, to

maintain lymph output (Carey, Small, & Bliss, 1983; Chang

& Bodmeier, 1997; Shah, Carvajal, Patel, Infeld, & Malick,

1994). Hauss, Fogal, and Ficorilli (1998) have developed a

rat model to allow collection of mesenteric lymph for 5 days

from conscious, minimally restrained animals with fully

patent cannulae and no signs of physical distress. This

model obviates the need for total restraint or general

anesthesia, both of which are known to influence intestinal

lymphatic transport of test compounds in unpredictable

ways. Animals were provided free access to an electrolyte

solution, which they consume in sufficient quantity to

maintain adequate lymph output without the need for the

previously required infusions for fluid replacement. How-

ever, the main limitation of this model is that both the

thoracic lymph duct and mesenteric lymph duct could not be

cannulated simultaneously.

The triple cannulated anaesthetized rat model (where the

mesenteric lymph duct, jugular vein, and duodenum are

accessed) has been used for the assessment of lymphatic

transport. General anesthesia precludes oral dosing in the

anaesthetized model, and consequently drug and lipid for-

mulations are administered intraduodenally (Charman &

Porter, 1996; Porter & Charman, 1997; Porter & Charman,

2001; White et al., 1991). This limitation thus circumvents

the inherent emulsifying action of the stomach and the

potential effects of lipids on gastric emptying. Thus, the

conscious rat model best represents the in vivo situation in

terms of both lack of anaesthetic effects and the ability to

orally administer drug formulations. In addition, in this

model both the thoracic lymph duct and mesenteric lymph

duct could not be cannulated simultaneously.

This paper describes the development of a cannulated rat

model, where both the thoracic and mesenteric lymph ducts

are accessed simultaneously for the assessment of drug

transport by the lymphatics and what percentage of lym-

phatic transport is a result of only intestinal lymphatics.

2. Methods

The animals were cared for in accordance with the

principles and guidelines of the Canadian Council on Animal

Care (CCAC). Male Sprague–Dawley rats (Charles River,

Montreal, Canada), 350–375 g in weight, were used for all

surgical procedures. The rat is the appropriate experimental

animal to investigate oral absorption and lymphatic transport

because intestinal characteristics (i.e., anatomical, metabolic,

and biochemical characteristics) of these animals are similar

to those found in humans (Fagerholm, Johansson, & Len-

nermas, 1996; Kararli, 1995; Levet-Trafit et al., 1996; Soria

& Zimmerman, 1996). Specifically, the intestinal processing

and absorption of dietary lipids are similar in rats and

humans (Pahl, Oveisi, Khamiseh, & Vaziri, 1998).

2.1. Surgical procedures

2.1.1. Mesenteric lymph duct cannulation (Fig. 1)

1. One hour preoperatively, the rat was given 1.5–2 ml of

olive oil by oral gavage for a 350- to 375-g rat.

M. Boyd et al. / Journal of Pharmacological and Toxicological Methods 49 (2004) 115–120 117

2. The rat was given 0.03–0.05 mg/kg atropine subcuta-

neously 20 min prior to surgery.

3. Induction of anesthesia was achieved with a 1-l oxygen

flow containing 5% isoflurane in a chamber.

4. The rat was transferred to a nose cone and a 2%

isoflurane solution with a 1-l oxygen flow was used to

maintain anesthesia during surgery.

5. Intravenous access was achieved using a 25-g butterfly

into the right tail vein. The butterfly was attached to a

60 drops/ml intravenous solution set and a 50-ml 0.09%

saline bag.

6. The rat was positioned in dorsal recumbency and hair

was clipped from the manubrium to the femoral area.

7. Upon removal of the hair, ophthalmic ointment was

applied generously to the eyes.

8. A three-step surgical scrub was preformed on the shaven

sites:

(a) First, the shaven sites are cleaned with a 0.5% P/V

chlorhexidine alcohol-based solution.

(b) Second, Betadine surgical scrub solution were ap-

plied with a clean 4� 4 in ever-increasing circles

away from midline. The removal of the scrub

solution follows a similar pattern with a sterile 4� 4.

(c) Third, the shaven areas were painted with a generous

layer of Betadine Solution.

9. A midline abdominal incision was made approximately

two thirds of the length of the abdomen posterior to the

xiphoid cartilage.

10. The intestine was brought out and wrapped in gauze that

has been soaked in warm saline.

11. The intestine was placed on the right side of the abdomen.

This exposes the posterior vena cava and the left renal

vein.

12. The mesenteric lymph duct was seen in the mesentery as

a white duct accompanying the mesenteric artery (see

Fig. 1). Both are attached with connective tissue.

13. Slight tension was placed on the duct and artery using

cotton tipped applicators, mall probes, and iris forceps.

Fig. 1. Retracted mesenteric lymphatic duct.

14. The connective tissue around the duct and artery was

cleared. A length of approximately 10 mm was suffi-

cient for cannulation.

15. When separated, vessel loops are placed around the

artery for retraction.

16. Two 4-0 silk ligatures are placed around the duct.

17. A 15-cm length of polyethylene tubing (id 0.58 mm, od

0.965 mm) was cut at one end diagonally to produce a

beveled point.

18. The tubing was primed with heparinized saline (20 units

heparin per ml). A syringe filled with heparinized saline

was left attached to the tubing.

19. A 16-G catheter was tunneled under the vena cava at the

point of angle that the lymph duct makes with the vena

cava. The catheter should be parallel and on top of the

duct.

20. The tubing was passed through the catheter and the

beveled tip is placed in position with the duct.

21. The catheter was removed.

22. To facilitate placement of the tubing into the duct, an

introducer was shaved at the tip to produce a more

obtuse angle and sharper tip.

23. Using the silk ties, gentle tension was applied and the

introducer was used to puncture the duct. With the

introducer in place, the tip of the tubing was slid into

the duct approximately 5 mm.

24. The syringe was detached from the tubing. Small drops

of heparinized saline are produced on the end of the

tubing as the lymph replaces the saline in the tubing.

25. Silk ligatures are tied down to secure tubing. Lymph

flow was seen from as a white solution in the tubing. If

a proper seal cannot be achieved with the ties, tissue

adhesive may be applied over the ligatures.

26. Warm saline was used to moisten the intestine and

abdominal cavity. The intestine was replaced in the

normal position.

27. Using continuous suturing, the peritoneum was closed

with a 4-0 vicryl taper needle. The skin was closed with

interrupted mattress sutures using a 4-0 vicryl cutting

needle.

28. Topical anesthetic cream was applied to the suture line

on the rat. Temgesic (buprenorphine, Schering-Plough)

was administered subcutaneously in the dose of 0.01–

0.05 mg/kg. This drug is commonly used immediately

after surgery to relieve pain associated with the surgery.

Emla cream (lidocaine 2.5% and prilocaine 2.5%) was

used topically to prevent itching. The rat was revived on

1 l oxygen and 0% isoflurane.

29. Oral gavage was preformed to administer the drug.

30. The rat remains warm in dorsal recumbency and kept

warm with IV fluids to promote good lymph flow.

Animals were under slight isoflurane anesthesia (1 l

oxygen flow containing 0.25% of isoflurane solution)

during lymph collection for 6–8 h.

31. Upon completion of lymph collection (6–8 h), 5 ml of

blood was collected by cardiac puncture and the rat was

Fig. 2. Cannulated thoracic lymphatic duct.

Fig. 3. Postoperation cannulated animal set-up.

M. Boyd et al. / Journal of Pharmacological and Toxicological Methods 49 (2004) 115–120118

euthanized with sodium pentobarbitol 120 mg/kg iv.

The right kidney, spleen, liver, heart, and lungs were

harvested.

2.1.2. Thoracic lymph duct cannulation (Fig. 2)

1. A 15-cm length of polyethylene tubing (id 0.58 mm, od

0.965 mm) was curved at one end by passing a bent wire

through it and plunging the curved end in boiling water

to produce a 2-cm end bent 160 relative to the rest of the

tubing. The tubing the gas sterilized (Fig. 2).

2. One hour preoperatively, the rat was given 1.5–2 ml of

olive oil by oral gavage for a 350- to 375-g rat.

3. The rat was given 0.03–0.05 mg/kg atropine subcuta-

neously 20 min prior to surgery.

4. Induction of anesthesia was achieved with a 1-l oxygen

flow containing 5% isoflurane in a chamber.

5. The rat was transferred to a nose cone, and a 2%

isoflurane solution with a 1-l oxygen flow was used to

maintain anesthesia during surgery.

6. Intravenous access was achieved using a 25-g butterfly

into the right tail vein. The butterfly was attached to a

60 drops/ml iv solution set and a 50 ml 0.09% saline

bag.

7. The rat was positioned in dorsal recumbency and hair

was clipped from the manubrium to the femoral area.

8. Upon removal of the hair, ophthalmic ointment was

applied generously to the eyes.

9. A three-step surgical scrub was preformed on the shaven

sites:

(a) First, the shaven sites were cleaned with a 0.5% P/V

chlorhexidine alcohol-based solution.

(b) Second, Betadine surgical scrub solution was applied

with a clean 4� 4 in ever increasing circles away

from midline. The removal of the scrub solution

follows a similar pattern with a sterile 4� 4.

(c) Third, the shaven areas were painted with a generous

layer of Betadine Solution.

10. A midline abdominal incision was made approximately

two thirds of the length of the abdomen posterior to the

xiphoid cartilage.

11. The intestine was brought out and wrapped in gauze that

has been soaked in warm saline.

12. The left kidney and adrenal were separated from the

connective tissue and fat. Together, kidney, adrenal, in-

testine, stomach, and spleenwere wrapped in warm saline

soaked gauze and retracted to the right side of the rat.

13. The thoracic lymph duct, lying parallel and partially

underneath the abdominal aorta, was separated away

from the vessel by applying slight pressure to the aorta

and gently teasing away connective tissue.

14. Vessel loops were placed around the abdominal aorta for

retraction.

15. Two 4-0 silk ligatures were placed around the lymph

duct.

16. The 15-cm length of polyethylene tubing was cut at the

bent end diagonally to produce a beveled point.

17. The tubing was primed with heparinized saline (20 units

heparin per ml). A syringe filled with heparinized saline

was left attached to the tubing.

18. A 16-G catheter was tunneled through the peritoneum

and skin in the lower left quadrant.

19. The tubing was passed through the catheter and the

beveled tip is placed in position with the duct.

20. The catheter was removed leaving the polyethylene

tubing in place.

21. To facilitate placement of the tubing into the duct, an

introducer was shaved at the tip to produce a more

obtuse angle and sharper tip.

22. Using the silk ties, gentle tension is applied and the

introducer was used to puncture the duct. With the

introducer in place, the tip of the tubing was slid into

the duct approximately 5 mm.

23. Silk ligatures were tied down to secure tubing. Lymph

flow was seen as a white solution in the tubing. If a

Fig. 4. Mesenteric lymphatic duct branches in 45% of Sprague–Dawley

rats used.

M. Boyd et al. / Journal of Pharmacological and Toxicological Methods 49 (2004) 115–120 119

proper seal cannot be achieved with the ties, tissue

adhesive may be applied over the ligatures.

24. The syringe was detached from the tubing. Small drops

of heparinized saline were produced on the end of the

tubing as the lymph replaces the saline in the tubing.

25. Warm saline was used to moisten the intestine and

abdominal cavity.

26. The intestine was replaced in the normal position. Using

continuous suturing, the peritoneumwas closed with a 4-

0 vicryl taper needle. The skin was closed with inter-

rupted mattress sutures using a 4-0 vicryl cutting needle.

27. Topical anesthetic cream was applied to the suture line

on the rat.

28. The rat was revived on 1 l oxygen and 0% isoflurane.

29. Oral gavage was preformed to administer the drug.

30. The rat remains warm in dorsal recumbency and kept

warm with IV fluids to promote good lymph flow.

31. Upon completion of lymph collection (6–8 h), 5 ml of

blood was collected by cardiac puncture and the rat was

euthanized with sodium pentobarbitol 120 mg/kg iv.

The right kidney, spleen, liver, heart, and lungs were

harvested (Fig. 3).

For both the mesenteric and thoracic surgical procedures,

the following information should be noted:

(a) The exteriorized cannula was placed underneath the

sterilized gauze.

(b) The two procedures (thoracic and mesenteric) were

initially carried out in different rats (to establish each

technique). However, several rats were done where both

the mesenteric and thoracic ducts were cannulated at the

same time.

(c) The animals were housed two per cage on a 12:12-h

light/dark schedule and given food (Purina lab chow)

and water ad libitum. Upon arrival, all animals were

allowed to acclimatize in their new surroundings for 10

days prior to surgery.

(d) Animals were under slight isoflurane anesthesia (1 l

oxygen flow containing 0.25% of isoflurane solution)

during lymph collection. In these initial studies, the

animals were not manually restrained or tethered using

a dual/multichanneled swivel mechanism.

3. Results and discussion

Following surgery, thoracic and mesenteric lymph flow

rates during the 24-h period immediately following surgery

averaged 12.5F 2.5 and 2.4F 1.1 ml/h, respectively. This

flow rate is greater than that obtained with previously

described methods, which require restraint of the animals

and/or a 24-h recovery period and are reported to produce

average intestinal lymph flow rates of 2 ml/h.

The use of our modified lymph model and the described

procedures, a skilled animal surgeon can expect a success rate

of approximately 80%. The main reasons for failure of the

model are dislodged, leaking, or improperly placed cannulae.

A dislodged or leaking cannula is suspected when the lymph

flow out of the animal is sluggish; but when the cannula is

raised, the lymph flows readily back into the animal. Another

issue not mentioned in previous research (Hauss, Fogal, &

Ficorilli, 1998; Hauss et al., 1998) is the physiology of the

mesenteric lymph duct. In approximately 45% of the rats

used in this study, the mesenteric lymph duct was multiply

branched making it next to impossible to cannulate it (Fig. 4).

This is the case for not only Sprague–Dawley rats but also

Long–Evans and Wistar rats (data not shown). This physio-

logic inconsistency is one limitation to this model, and

scientists should be aware of when initiating this surgical

procedure.

In conclusion, cannulation of the thoracic and mesenteric

lymph ducts within the rat has been successfully developed

for the assessment of drug transport through the lymphatics.

This model could be utilized to evaluate the role of

lymphatics in assessing the bioavailability of drugs

incorporated into novel lipid-based formulations.

Acknowledgements

This study was supported with an operating grant from

the Canadian Institutes of Health Research (Grant #MOP-

49432 to KMW).

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