Journal of Pharmacological and Toxicological Methods 61 (2010) 2026

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Journal of Pharmacological and Toxicological Methods

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j p h a r m t ox

Original article

A comparative study of four permanent cannulation procedures in rats

Luka Peternel , pela krajnar, Manica erne
Lek Pharmaceuticals d.d., Verovkova 57, 1529 Ljubljana, Slovenia

a r t i c l e i n f o a b s t r a c t

Article history: Introduction: Permanent implantation of vascular catheters allows multiple blood sampling from the
Received 20 April 2009 experimental animal. The aim of the study was rstly to establish four cannulation procedures in rats, which
Accepted 10 July 2009 will result in high rate of catheter patency after the post surgical recovery period and secondly to determine a
suitability of food and water consumption (FC and WC) and body weight gain (BW) as markers for the
Keywords:
estimation of the length of recovery period. Methods: Cannulation of the jugular vein (n = 12), femoral vein
Cannulation
(n = 15), ileocolic vein (n = 13) and the dual cannulation of the jugular vein/duodenum (n = 7) were
Catheter
Post surgical recovery
performed in the rat. Catheter patency was monitored throughout the recovery period. Rats were considered
Rat fully recovered after return of BW, FC and WC to the preoperative values. Results: A decrease of catheter
Vascular surgery patency in the recovery period followed a linear regression model in all cannulation groups (P b 0.01,
Methods R2 N 0.87). BW signicantly decreased in the rats subjected to ileocolic and jugular/duodenum cannulation
procedures only. A signicant transient decrease of FC and increase of WC was observed in all cannulation
groups. FC returned to the preoperative values more slowly than BW and WC. Therefore FC was considered as
the most sensitive parameter for the estimation of the length of recovery period, resulting in the recovery
period of 10 days and catheter patency of 7585% in the case of jugular vein, femoral vein and dual jugular
vein/duodenum cannulation procedures. After the cannulation of the ileocolic vein FC did not return to the
preoperative values. Discussion: The catheter patency at the end of recovery period, which was estimated by
the FC, was assured in the majority of rats in all cannulation groups, with the exception of the ileocolic vein
cannulation group. In this particular cannulation group FC did not return to preoperative values, thus
indicating that further optimization of the ileocolic vein cannulation procedure is required.
2009 Elsevier Inc. All rights reserved.

1. Introduction
An appropriate pharmacokinetic (PK) prole remains a hurdle to
reducing risk and improving productivity in pharmaceutical research
and development (Alavijeh & Palmer, 2004; Roberts, 2003). A number
of new in silico, in vitro and in vivo techniques are available to screen
compounds for key absorption, distribution, metabolism and excre-
tion (ADME) characteristics, which, when applied within a rational
strategy, can make a major contribution to the design and selection of
successful new chemical entities (Alavijeh & Palmer, 2004; Roberts,
2003).
A rat has been a useful laboratory animal in virtually every area of
biomedical research, including the ADME and PK studies. The study of
drug PK in the rat is solely dependent upon procedures that allow
blood sampling. Blood can be sampled from the rat in a number of
ways, for example by puncturing the tail vein (van Herck et al., 2001),
the sublingual vein (Zeller, Webber, Panoussis, Brge, & Bergmann,
1998) and the saphenous vein (Hem, Smith, & Solberg, 1998) or by
using implanted vascular catheters (Hall, Ross, Bozovic, & Grant, 1984;
Corresponding author. Tel.: +386 15803315; fax: +386 15682340.
E-mail address: luka.peternel@sandoz.com (L. Peternel).
Remie, van Dongen, Rensema, & van Wunnik, 1990; Thrivikraman,
Huot, & Plotsky, 2002). Each method has its drawbacks and limitations,
which should be identied in order to minimize the impact of these
drawbacks on experimental results. For example, the sampling method
can inuence clinical pathology parameters (Schnell, Hardy, Hawley,
Propert, & Wilson, 2002; Upton & Morgan 1975), renal functional
parameters (Verbaeys, Ringoir, van Maele, & Lameire, 1995), beha-
vioral variables (van Herck et al., 2001) and pharmacokinetics of drugs
(Hui et al., 2007). In the case of intravenous PK studies, a preferred
method to obtain multiple blood samples is a chronically implanted
vascular catheter, for which the surgical procedure is required (Hui et
al., 2007; Wiersma & Kastelijn 1985). A number of different surgical
techniques have been developed over the past decades, as for example
cannulation of the jugular vein (Remie et al., 1990; Thrivikraman et al.,
2002), femoral vein (Hall et al., 1984) and portal vein (Remie et al.,
1990; Tordoff, Schulkin, & Friedman, 1986). The development of these
techniques has allowed researchers to incorporate permanently
cannulated rats as a powerful tool in biomedical research. However,
as important as surgical techniques per se, is careful post surgical
monitoring in order to perform the experiments in fully recovered rats.
Only then the experiments could be considered as scientically and
ethically justied. In contrast to radio-telemetry related surgery
procedures (Handoko et al., 2008; Kaidi et al., 2007; Sharp, Zammit,

1056-8719/$ see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.vascn.2009.07.004
 L. Peternel et al. / Journal of Pharmacological and Toxicological Methods 61 (2010) 2026
Azar, & Lawson, 2003), a detailed post surgical recovery data and
catheter patency after vascular cannulation procedures in rats are
sparse.
In the present study we evaluated the permanent cannulation of
the femoral vein, jugular vein and ileocolic vein and the dual
cannulation of the jugular vein/duodenum in the rat. Detailed surgical
procedures are described herein. In addition to the catheter patency, a
suitability of food and water consumption (FC and WC) and body
weight gain (BW) as markers for the estimation of the length of
recovery period after each cannulation procedure is disclosed.
2. Materials and methods
2.1. Statement on use and care of animals
Studies were carried out using male Wistar rats (265349 g, Lek
Pharmaceuticals d.d., Slovenia) with good conventional microbial
status. Before surgery the rats were housed 35 per cage. The room
temperature was kept at 23 1 C, humidity at 4070% and a 12 h
light dark cycle was applied. Food and water were available ad libitum.
All animals received care in compliance with the European Conven-
tion on Animal Care and were approved by the local regulatory
authorities.
2.2. Surgical procedures
2.2.1. Preparation of the rat for surgery
Rats were anaesthetized with intraperitoneal injection of a
mixture of ketamine (Bioketan, Vetoquinol Biowet, Poland), xylazine
(Chanazine, Chanelle Pharmaceuticals Manufacturing Ltd., Ireland)
and saline (5:4:5 v/v/v; 0.7 ml/rat, 20% w/v solution). If a prolongation
of anaesthesia during the operation was required, rats were subjected
to 2% isouorane (Isouran, Nicholas Piramal India LTD, Alpine Hous,
England) in oxygen (1.5 L/min) inhalation anaesthesia. Once anaes-
thetized, an ocular lubricant Bivacyn (Lek Pharmaceuticals d.d.,
Slovenia) was applied to both of the animal's eyes. All incision sites
were shaved and disinfected with an iodine solution (Iodine Povidon,
3.75% in saline, Pharmacy of Ljubljana Medical Centre, Slovenia). The
rat was then positioned on a sterile operation cover, which was placed
over a homeothermic heated plate (37 C) with a temperature
feedback loop (Harvard Apparatus, Germany). Finally, the incision site
was covered with a sterile drape.
2.2.2. Preparation of catheters
Rats were implanted with sterile catheters (autoclavable at 121 C,
20 min) made of RenaSil silicone tubes (0.09 cm OD 0.06 cm ID,
Braintree Scientic, USA). The jugular, femoral and duodenum catheters
were equipped with the silicone ring 3.5, 4.5 and 1 cm from the tip of the
catheter, respectively. Ileocolic catheters were prepared without a
silicone ring. The end of the catheters inserted into a vein and duodenum
were slightly beveled to facilitate the catheter insertion.
2.2.3. Surgical procedures
All surgical procedures were performed with sterile surgical set
(autoclavable at 121 C, 20 min) by using dissecting microscope OPMI
Pico (Zeiss, Germany). With the exception of abdominal muscles closure,
silk ligatures were used in all surgical procedures. Excellent handling
properties and good knot security dictated the choice for silk suture
material. At necropsy we did not observed any signs of irritation or
inammation at the sites where the ligatures were put around the
vessels. All exposed tissues were continuously moistened in order to
prevent dehydration, particularly in the case of abdominal surgical
procedures. The blood loss during the surgery procedures was negligible.
2.2.3.1. Cannulation of jugular vein. We used a modied technique of
cannulating the jugular vein proposed by Thrivikraman et al. (2002).
21
A 2 cm longitudinal incision was made just above the right clavicle.
The jugular vein was exposed and freed from the adhering tissue. Two
4-0 ligatures (Silkam, Braun, Germany) were passed under the vein
and a cranial ligature was tied to stop blood ow. The free ends of a
cranial ligature were clamped with a hemostat and a vein was gently
stretched. The second ligature was placed caudally and tied with one
loose knot. With caution a stab wound was made with the 25 G needle
into the upper surface of the vein and the beveled end of the catheter
connected to a 1 ml syringe lled with heparinized saline (50 IU
heparin/ml, Krka, Slovenia) was slid into the vein. The catheter was
advanced up to the silicone ring and tied to the vein with a cranial
ligature to hold it in place. 500 l of heparinized saline (50 IU heparin/
ml, Krka, Slovenia) was slowly injected through the catheter, and then
a slow withdrawal of blood was attempted. If there was any resistance,
the catheter was repositioned by minimal advancement or retraction
until blood was easily withdrawn. After patency was established, the
catheter was tied to the vein with a caudal ligature. The free ends of
the caudal and cranial ligature were positioned over the silicone ring
and tied together. The catheter was tunneled subcutaneously through
the trocar to the intrascapular region and the incision wound was
closed with wound clips.
2.2.3.2. Cannulation of the femoral vein. We used a modied technique
of cannulating the femoral vein proposed by Hall et al. (1984). The 1.5
2 cm long incision was made in the skin proximal to the right inguinal
region. Connective tissue was teased away and the femoral vein was
gently separated from the femoral artery and femoral nerve. Two 4-0
ligatures (Silkam, Braun, Germany) were passed under the vein. The
caudal ligature was tied around the vein to stop the blood ow and the
proximal ligature was tied with one loose knot. The free ends of both
ligatures were clamped with a hemostat and the vein was gently
stretched. After a stab wound was made with the 25 G needle, the
catheter insertion, patency check and subcutaneous tunneling of the
catheter were performed in the same manner as described for the
cannulation of the jugular vein.
2.2.3.3. Cannulation of the ileocolic vein. We used a modied technique
of cannulating the ileocolic vein proposed by Tordoff et al. (1986). The
abdomen was entered through a midline incision, and the caecum was
gently lifted out of the peritoneum onto sterile gauze moistened with
warm saline. The main branch of the ileocolic vein, which travels between
the caecum and liver, was located, and the mesentery around the junction
of this vessel and one of its tributaries close to the caecum was gently
cleared of fat. The rst 4-0 ligature (Silkam, Braun, Germany) was placed
caudally around the ileocolic vein and tight, the second ligature was
placed cranially around the ileocolic vein and the third ligature was
placed around the tributary vein and they were both tied with loose
knots. The free ends of all ligatures were clamped with hemostats and the
ileocolic vein was gently stretched. A stab wound was made with a 25 G
needle at the point where the tributary joined the main branch of the
ileocolic vein and the beveled end of the catheter connected to a 1 ml
syringe, which was lled with heparinized saline (50 IU heparin/ml, Krka,
Slovenia), was gently slid into the vein and the catheter was threaded
towards the liver up to the point at which a gentle pressure was detected.
The catheter was then retracted to a position where blood could be
withdrawn. At this point the catheter was secured with a proximal
ligature. After securing the catheter, 800 l of heparinized saline (50 IU
heparin/ml, Krka, Slovenia) was slowly injected through the catheter
followed by a slow attempt to withdraw blood. After catheter patency was
established, the ligature loop around the tributary vein was removed; the
catheter was secured in place by tying together the loose ends of the tied
ligatures. The caecum was carefully returned to its normal position within
the abdominal cavity. The catheter was tunneled subcutaneously through
the trocar to the intrascapular region. The abdominal muscles were closed
with a 6-0 absorbable suture (Sale, Braun, Germany) and the abdominal
skin was closed with wound clips.
22 L. Peternel et al. / Journal of Pharmacological and Toxicological Methods 61 (2010) 2026
2.2.3.4. Cannulation of the jugular vein and duodenum. Following
cannulation of the jugular vein as described above the abdomen was
entered through a midline incision, and the guts were gently torn apart
in order to locate the duodenum (Torres-Molina et al., 1996). The part of
the duodenum caudal to the pancreas was gently lifted. Four or ve
purse-string sutures (Premilene 6-0, Braun, Germany) were placed in
the duodenum wall. After each stitch any leaking was immediately
arrested by applying light pressure on the perforated area of the
duodenum. Using a 25 G needle the duodenum was perforated in the
middle of a purse-string. Subsequently the cannula lled with saline was
inserted into the duodenum and pushed in up to the silicone ring. The
purse-string was tightened and the drawstrings of the suture were used
to anchor the cannula behind the silicone ring. The duodenum was
carefully returned to its normal position within the abdominal cavity.
The catheter was tunneled subcutaneously through the trocar to the
intrascapular region. The abdominal muscles were closed with a 6-0
absorbable suture (Sale, Braun, Germany) and the abdominal skin was
closed over the catheter with wound clips.
2.2.4. Preparation of the head anchor
At the intrascapular region a 12 cm longitudinal incision was
made to facilitate the catheter exteriorization. The exteriorized end of
the catheter was attached to sterile stainless L-shaped adapter (Tik,
Slovenia), on which the adhesive tissue stimulating mesh (1 cm2,
Premilene, Braun, Germany) was glued with histoacryl (Braun,
Germany). The part of the L-shaped adapter with an attached mesh
and catheter was inserted subcutaneously and the wound was closed
with a 6-0 suture (Silkam, Braun, Germany). On the external part of
the L-shaped adapter a 0.5 cm long plug made of CO-EX tube (Instech
Solomon, USA) was attached and heat sealed. Prior to sealing, the
vascular catheters and duodenum catheter were lled with a lock
solution (60% v/v glycerol in saline, 500 IU heparin/ml; in the case of
the duodenum catheter heparin was not used). The incision was
closed with a 4.0 suture (Silkam, Braun, Germany).
2.2.5. Postsurgical procedures
After surgery, the rats were individually caged and placed in a warm
room (T = 26 1 C) for 24 h. Saline (3 ml per kg body weight) was
administered to each rat via intravenous infusion. The incision sites were
sprayed with Bivacyn (Lek Pharmaceuticals d.d., Slovenia) and 0.03 ml/
rat of antibiotic Trioxyl (Univet Ltd, Ireland, 150 mg/ml) was
administered intramuscularly. On days 1, 2, 3, 4, 7, 10, 14, 17 and 21
after the surgery, body weights (BW), food consumption (FC) and water
consumption (WC) were recorded. FC and WC were normalized on the
consumption per day. The length of post surgical recovery period was
estimated as a time required for return of BW, FC and WC to the
Fig. 1. Catheter patency after cannulation of the jugular vein (Jug, n = 12), femoral vein (Fem, n = 15), ileocolic vein (Ileo, n = 13) and jugular vein/duodenum (Jug/Duod, n = 7). Day 0
represent catheter patency (%) immediately after the surgery.
preoperative values, as judged by nonsignicant difference with regard
to preoperative (day 0) values (Handoko et al., 2008; Kaidi et al., 2007;
Sharp et al., 2003). The patency of catheters was checked twice per week
by the following procedure: removal of the old plug, putting on the new
plug, aspiration of the lock solution, in the case of the vascular catheters
aspiration of 200 l of the blood sample, injection of 200 l of saline,
lling the catheter with lock solution and heat sealing a new plug. The
number of white blood cells (WBC) was measured in the withdrawn
blood samples by using an automated blood counter ABC vet machine
(ABX Diagnostics, France). The wound clips and ligatures were removed
710 days after surgery. The removal of wound clips was well tolerated
by rats and light sedation was not necessary. On the other hand skin
ligatures were removed under light sedation with isouorane (Isouran,
Nicholas Piramal India LTD, Alpine Hous, England).
2.2.6. Statistical analysis
A signicant difference of FC, WC and BW with respect to
preoperative values within each cannulation group was determined
by repeated measures one-way ANOVA followed by a Dunnett post test.
The Greenhouse Geisser correction was applied, but repeated measures
one-way ANOVA statistics did not change, thus indicating that the
assumption of circularity was not violated. Data were additionally
analyzed by using linear regression model (Y =kX+n) or nonlinear
regression exponential model as indicated within the text. The equation
used for nonlinear regression exponential model was as follows:
Y = plateau+ (top-plateau) * (1e k(X Xo), where plateau is dened
as Ymin and Xo as X value where an exponential increase starts. P b 0.05
was considered to be signicant. All data are given as mean S.E.M.. The
statistical and graphical analysis was supported by the Graph Pad Prism
version 4.02 for Windows (Graph Pad Software, USA).
3. Results
3.1. Catheter patency
Fig. 1 shows catheter patency in a time-dependent manner for four
cannulation groups. In all groups a steady decrease of catheter patency
following simple linear regression model (P b 0.01, R2 N 0.87) was
observed. The percentage of the patent catheters at the end of the
recovery period is shown in Table 1. At the end of investigated period
catheter patency in the jugular, femoral and ileocolic cannulation
groups was 6 out of 12, 7 out of 15 and 7 out of 13, respectively. These
results remained constant between day 21 and 28 of the experiment,
what is desired with regard to chronic intravenous administration
procedures. On the other hand, in the jugular/duodenum cannulation
group the percentage of the patent catheters signicantly dropped in
 L. Peternel et al. / Journal of Pharmacological and Toxicological Methods 61 (2010) 2026 23

Table 1 the last 7 days of experiment, from 4 out of 7 (day 21) to 2 out of 7
Catheter patency at the end of recovery period. (day 28). As shown in Table 1, catheter patency at the end of recovery
BW FC WC period was retained in the majority of catheters in the jugular, femoral
Cannulation Recovery Catheter Recovery Catheter Recovery Catheter and jugular/duodenum cannulation groups, but not in the ileocolic
group period patency period patency period patency cannulation group.
(day) (%) (day) (%) (day) (%)
Jugular vein 1 100 10 75 7 92 3.2. Body weight (BW)
(n = 12)
Femoral vein 1 100 10 80 4 87
(n = 15)
In the jugular and femoral vein cannulation groups a signicant
Ileocolic vein 7 77 N21 53 4 77 drop of BW in comparison to preoperative values was not observed.
(n = 13) On the other hand BW signicantly decreased in the rats subjected to
Jugular vein/ 10 85 10 85 7 85 ileocolic and jugular/duodenum cannulation procedures, followed by
duodenum
its return to the preoperative values on day 7 (ileocolic) and 10
(n = 7)
(jugular/duodenum) of the experiment (Fig. 2). With the exception of
The length of recovery period was estimated by the return of body weight (BW), food
jugular/duodenum cannulation group a signicant increase of BW
consumption (FC) or water consumption (WC) to the preoperative values. n, number of
subjects.
with respect to day 0 was observed after day 10 (jugular group) and
after day 14 of the experiment (femoral and ileocolic groups). A
pattern of BW increase followed simple linear regression model in the

Fig. 2. Body weight (BW; upper graph), food consumption (FC; middle graph) and water consumption (WC; lower graph) after cannulation of the jugular vein (Jug, n = 12), femoral
vein (Fem, n = 15), ileocolic vein (Ileo, n = 13) and jugular vein/duodenum (Jug/Duod, n = 7). Data are shown as mean S.E.M., RM ANOVA, *P b 0.01, #P b 0.05 with respect to
preoperative values (day 0).
24 L. Peternel et al. / Journal of Pharmacological and Toxicological Methods 61 (2010) 2026
jugular (P b 0.01, R2 = 0.64), femoral (P b 0.01, R2 = 0.60) and ileocolic
(P b 0.01, R2 = 0.74) cannulation groups. Although linear relationship
was considered signicant also in the jugular/duodenum cannulation
group (P b 0.01), goodness of t of was poor (R2 = 0.23).
3.3. Food consumption (FC)
A signicant decrease of FC in comparison to preoperative values was
observed in all cannulation groups (Fig. 2). In the jugular, femoral and
jugular/duodenum cannulation groups FC returned to preoperative
values on day 10 of the experiment. Such a pattern was not observed in
the ileocolic cannulation group, where a signicant decrease of FC with
respect to preoperative values was observed at all time points.
The mode of FC increase followed a simple linear regression model in
the jugular (P b 0.01, R2 =0.46), femoral (P b 0.01, R2 = 0.42), ileocolic
(P b 0.01, R2 = 0.39) and jugular/duodenum (P b 0.01, R2 = 0.54) cannula-
tion groups.
3.4. Water consumption (WC)
WC was transiently signicantly increased in all 4 cannulation
groups, resulting in a maximum WC on day 3 (femoral and ileocolic) and
on day 4 (jugular and jugular/duodenum) of the experiment (Fig. 2). The
pattern of WC did not follow the linear regression model in any of the
investigated cannulation procedure and data were scattered.
3.5. White blood cells (WBC)
The number of WBC remained within threshold values (315 109/L)
in all cannulation groups throughout the experiment. This indicates that
rats were not subjected to systemic infection.
4. Discussion
The purpose of this work was to evaluate four different cannulation
procedures which can be used in multiple blood sampling experi-
Fig. 3. Association between food consumption (FC) and body weight (BW) as described by nonlinear regression exponential model (jugular, femoral and ileocolic vein cannulation
groups) or linear regression model (jugular/duodenum cannulation group). Dotted lines represent 95% condence interval of the regression model (solid line). Jug, jugular; Fem,
femoral; Ileo, ileocolic; Jug/Duod, jugular/duodenum.
ments in the rat, as for example in ADME and PK studies (Aghazadeh-
Habashi, Ibrahim, Carran, Anastassiades, & Jamali, 2006; Matsumoto,
Chiji, & Hara, 2005; Shayeganpour, Hamdy, & Brocks, 2007; Wan,
Ernstgrd, Song, & Shoaf, 2006). As outlined in our study catheter
patency and recovery period are inversely proportioned, what makes
procedures with short post surgical recovery periods superior over the
others. Prolonged recovery periods increase the chance for catheter
failure probably due to sleeve of collagen, cells and brin formed
around the intravascular catheter (Forauer, Theoharis & Dasika, 2006;
Xiang, Verbeken, Lommel, Stas, & Wever, 1998; Yang, Maarek, &
Holschneider, 2005).
The key question is by which parameter (BW, FC or WC) the length of
recovery period can be most reliably assessed. Within particular
cannulation group usage of BW, FC or WC produced signicant
variations in the estimation of the length of recovery period (Table 1).
For example, in the jugular and femoral vein cannulation groups a
decrease of BW was not considered signicant, but a signicant drop of
FC was nevertheless noticed, resulting in marked differences in the
estimation of the length of recovery period. Such discrepancies were
addressed by modeling the association between BW, FC and WC. An
association among BW and FC followed a nonlinear exponential model
in the jugular, femoral and ileocolic cannulation procedures, but a
steeper rise to the plateau was determined in the jugular and femoral
vein cannulation groups only (Fig. 3). This observation indicates that in
the case of jugular and femoral vein cannulation group small BW
changes resulted in large FC changes, although causality for this effect
was not established. In the case of ileocolic and jugular/duodenum
cannulation groups the time to reach the plateau is prolonged (Fig. 3),
what is in line with signicant decrease of both BW and FC. In the
ileocolic cannulation group FC is signicantly decreased even at the end
of investigated period despite signicant increase of BW, which probably
indicates an appetite decrease effect caused by the cannulation of the
ileocolic vein. In contrast to others in the jugular/duodenum cannulation
group the association between FC and BW followed the linear regression
model even better than nonlinear exponential model. No meaningful
association was observed between WC and BW or between WC and FC in
 L. Peternel et al. / Journal of Pharmacological and Toxicological Methods 61 (2010) 2026
any cannulation group, but the estimation of the length of recovery
period based on WC is somehow shorter. It should be also noted that
large variations were observed in the measurement of this parameter
which hampered its use as a reliable marker for the estimation of post
surgical recovery period. An increase need of water intake in the rst few
days after surgery was also observed in all cannulation groups. This
could indicate a transient dehydration of the rats that resulted in an
increase need for uid intake (Sanders et al., 2001; Sigurdsson 1995). In
addition to carefull surgical procedures and consequently negligible
blood loss, intensive tissue moistening during the surgeries and infusion
of saline at the end of surgical procedures more intensive uid
replacement might decrease a need for increase post surgical water
intake. To sum up, BW, FC and WC should all return to preoperative
values to consider rat as fully recovered from the surgery. As judged from
Table 1, FC returned to preoperative values more slowly than BW and WC
and as such could be considered as the most sensitive parameter for the
estimation of the length of recovery period.
It is generally agreed that effective postsurgical analgesia care results
in more rapid recovery and reduction of distress (Stokes, Flecknell, &
Richardson, 2009). However, some commonly used analgesics can
induce physiological responses which may actually slow the recovery
process. For example, buprenorphine and butorphanol delayed the
return of food intake to presurgical levels compared with rats receiving
neither analgesic (Jablonski, Howden, & Baxter, 2001; Sharp et al., 2003),
while administration of ketoprofen did not shorten the return of food
intake to presurgical levels (Sharp et al., 2003). In another study
ketorolac slow down the body weight increase in rats subjected to
laparotomy (Welberg et al., 2006). Probably, analgesics are effective in
relieving pain but had pharmacological side effects that can even
prolong the recovery period. In order to avoid undesired impact of
analgesics on measured parameters (BW, FC and WC) and consequently
on the estimation of the length of recovery period supplementary
analgesia treatment was not used in the present study. This decision was
justied by the fact, that anaesthesia mixture used in the present study
possess analgesic effect (Abbott & Bonder, 1997; Bell, Dahl, Moore, &
Kalso, 2005; Welberg et al., 2006). In particular ketamine can be also
used for the treatment of postoperative pain at subanaesthetic doses (De
Kock & Lavand'homme, 2007). Moreover, in a pilot study rats were cage
monitored during the recovery period for clinical signs related to pain,
which were, however, absent.
Easy access and detailed surgical procedures (Remie et al., 1990;
Steffens 1969; Thrivikraman et al., 2002; Upton 1975) established
cannulation of jugular vein as the most commonly used cannulation
site in intravenous rat PK studies (Bae et al., 2007; Chung et al., 2007;
Lee, Shin, Bae, & Lee, 2006). However, cannulation of the femoral vein
is a favored cannulation site in the case of long term continuous
intravenous infusion studies (Green & Patten 2000). In comparison to
the cannulation of the jugular vein and femoral vein, the cannulation
of the ileocolic vein is a time consuming surgical procedure (Braillon &
Brody 1988; Gallo-Torres & Ludorf 1974; Remie et al., 1990; Suzuki,
Saito, Isozaki, & Ishida, 1973; Tordoff et al., 1986). Nevertheless, by
cannulating the ileocolic vein, blood samples can be obtained from the
portal vein, which is a meaningful way to study drug absorption and to
differentiate between the intestinal and hepatic metabolism of new
drug candidates in vivo (Doherty & Pang 1997; Kullak-Ublick & Becker
2003; Paine & Oberlies 2007). Another rarely used model is the dual
cannulation of the jugular vein and duodenum in the same rat
(Torres-Molina et al., 1996). Because development of gastric resistant
formulation rarely takes place early in a drug discovery process, such a
model can be used to circumvent gastric degradation related issues,
while the systemic blood can be obtained at the same time.
In conclusion, more pretentious surgical procedures like cannula-
tion of the ileocolic vein resulted in prolonged post surgical recovery
period. In other cannulation groups the catheter patency is assured
throughout the post surgical recovery period in a majority of rats.
With regard to the markers used for the estimation of the length of
25
post surgical recovery period, FC was considered as the most sensitive
one. Based on FC data jugular, femoral and jugular/duodenum
cannulation procedures required the recovery period of 10 days,
while the cannulation of the ileocolic vein should be further optimized
in order to assure full recovered rats.
Acknowledgement
The authors would like to thank Tatjana Zajc for her excellent
technical support.
References
Abbott, F. V., & Bonder, M. (1997). Options for management of acute pain in the rat. The
Veterinary Record, 140, 553557.
Aghazadeh-Habashi, A., Ibrahim, A., Carran, J., Anastassiades, T., & Jamali, F. (2006).
Single dose pharmacokinetics and bioavailability of butyryl glucosamine in the rat.
Journal of Pharmacy & Pharmaceutical Sciences, 9, 359364.
Alavijeh, M. S., & Palmer, A. M. (2004). The pivotal role of drug metabolism and
pharmacokinetics in the discovery and development of new medicines. IDrugs: The
Investigational Drugs Journal, 7, 755763.
Bae, S. K., Yang, S. H., Shin, K. N., Rhee, J. K., Yoo, M., & Lee, M. G. (2007). Pharmacokinetics of
DA-7218, a new oxazolidone, and its active metabolite, DA-7157, after intravenous and
oral administration of DA-7218 and DA-7157 to rats. The Journal of Pharmacy and
Pharmacology, 59, 955963.
Bell, R. F., Dahl, J. B., Moore, R. A., & Kalso, E. (2005). Peri-operative ketamine for acute
post-operative pain: A quantitative and qualitative systematic review (Cochrane
review). Acta Anaesthesiologica Scandinavica, 49, 14051428.
Braillon, A., & Brody, M. J. (1988). A simple method for chronic cannulation of the portal
vein in intact unrestrained rats. The American Journal of Physiology, 255, G191193.
Chung, H. J., Lee, J. H., Woo, S. J., Park, H. K., Koo, C. H., & Lee, M. G. (2007). Pharmacokinetics
of L-FMAUS, a new antiviral agent, after intravenous and oral administration to rats:
Contribution of gastrointestinal rst-pass effect to low bioavailability. Biopharmaceutics
& Drug Disposition, 28, 187197.
De Kock, M. F., & Lavand'homme, P. M. (2007). The clinical role of NMDA receptor antagonists
for the treatment of postoperative pain. Baillire's best practice & research. Clinical
Anaesthesiology, 21, 8598.
Doherty, M. M., & Pang, K. S. (1997). First-pass effect: Signicance of the intestine for
absorption and metabolism. Drug and Chemical Toxicology, 20, 329344.
Forauer, A. R., Theoharis, C. G., & Dasika, N. L. (2006). Jugular vein catheter placement:
histologic features and development of catheter-related (brin) sheaths in a swine
model. Radiology, 240, 427434.
Gallo-Torres, H. E., & Ludorf, J. (1974). Techniques for the in vivo catheterization of the
portal vein in the rat. Proceedings of the Society for Experimental Biology and Medicine,
145, 249254.
Green, O. P., & Patten, D. (2000). Femoral cannulation using the jacket/harness model in
the rat. In G. Healing & D. Smith (Eds.), Handbook of pre-clinical continuous intravenous
infusion (pp. 719). London: Taylor & Francis.
Hall, R. I., Ross, L. H., Bozovic, M., & Grant, J. P. (1984). A simple method of obtaining repeated
venous blood samples from the conscious rat. The Journal of Surgical Research, 36,
9295.
Handoko, M. L., Schalij, I., Kramer, K., Sebkhi, A., Postmus, P. E., van der Laarse, W. J., et al.
(2008). A rened radio-telemetry technique to monitor right ventricle or pulmonary
artery pressures in rats: A useful tool in pulmonary hypertension research. Pgers
Archiv: European Journal of Physiology, 455, 951959.
Hem, A., Smith, A. J., & Solberg, P. (1998). Saphenous vein puncture for blood sampling of
the mouse, rat, hamster, gerbil, guinea pig, ferret and mink. Laboratory Animals, 32,
364368.
van Herck, H., Baumans, V., Brandt, C. J., Boere, H. A., Hesp, A. P., van Lith, H. A., et al.
(2001). Blood sampling from the retro-orbital plexus, the saphenous vein and the
tail vein in rats: Comparative effects on selected behavioural and blood variables.
Laboratory Animals, 35, 131139.
Hui, Y. H., Huang, N. H., Ebbert, L., Bina, H., Chiang, A., Maples, C., et al. (2007).
Pharmacokinetic comparisons of tail-bleeding with cannula- or retro-orbital bleeding
techniques in rats using six marketed drugs. Journal of Pharmacological and
Toxicological Methods, 56, 256264.
Jablonski, P., Howden, B. O., & Baxter, K. (2001). Inuence of buprenorphine analgesia
on post-operative recovery in two strains of rats. Laboratory Animals, 35, 213222.
Kaidi, S., Brutel, F., van Deun, F., Kramer, K., Remie, R., Dewe, W., et al. (2007). Comparison of
two methods (left carotid artery and abdominal aorta) for surgical implantation of
radiotelemetry devices in CD-1 mice. Laboratory Animals, 41, 388402.
Kullak-Ublick, G. A., & Becker, M. B. (2003). Regulation of drug and bile salt transporters
in liver and intestine. Drug Metabolism Reviews, 35, 305317.
Lee, D. Y., Shin, H. S., Bae, S. K., & Lee, M. G. (2006). Effects of enzyme inducers and inhibitors on
the pharmacokinetics of intravenous omeprazole in rats. Biopharmaceutics & Drug
Disposition, 27, 209218.
Matsumoto, M., Chiji, H., & Hara, H. (2005). Intestinal absorption and metabolism of a
soluble avonoid, alphaG-rutin, in portal cannulated rats. Free Radical Research, 39,
11391146.
Paine, M. F., & Oberlies, N. H. (2007). Clinical relevance of the small intestine as an organ
of drug elimination: Drugfruit juice interactions. Expert Opinion on Drug
Metabolism & Toxicology, 3, 6780.
26 L. Peternel et al. / Journal of Pharmacological and Toxicological Methods 61 (2010) 2026
Remie, R., van Dongen, J. J., Rensema, J. W., & van Wunnik, G. H. J. (1990). Manual of
microsurgery on the laboratory rat. In J. P. Huston (Ed.), Techniques in the behavioral
and neural sciences, Volume 4 (pp. 1293). London: Elsevier.
Roberts, S. A. (2003). Drug metabolism and pharmacokinetics in drug discovery. Cur-
rent Opinion in Drug Discovery & Development, 6, 6680.
Sanders, G., Mercer, S. J., Saeb-Parsey, K., Akhavani, M. A., Hosie, K. B., & Lambert, A. W.
(2001). Randomized clinical trial of intravenous uid replacement during bowel
preparation for surgery. The British Journal of Surgery, 88, 13631365.
Schnell, M. A., Hardy, C., Hawley, M., Propert, K. J., & Wilson, J. M. (2002). Effect of blood
collection technique in mice on clinical pathology parameters. Human Gene Therapy,
13, 155161.
Sharp, J., Zammit, T., Azar, T., & Lawson, D. (2003). Recovery of male rats from major
abdominal surgery after treatment with various analgesics. Contemporary Topics in
Laboratory Animal Science, 42, 2227.
Shayeganpour, A., Hamdy, D. A., & Brocks, D. R. (2007). Pharmacokinetics of
desethylamiodarone in the rat after its administration as the preformed metabolite,
and after administration of amiodarone. Biopharmaceutics & Drug Disposition, 29,
159166.
Sigurdsson, G. H. (1995). Perioperative uid management in microvascular surgery.
Journal of Reconstructive Microsurgery, 11, 5765.
Steffens, A. B. (1969). A method for frequent sampling of blood and continuous infusion
of uids in the rat without disturbing the animal. Physiology & Behavior, 4,
833836.
Stokes, E. L., Flecknell, P. A., & Richardson, C. A. (2009). Reported analgesic and
anaesthetic administration to rodents undergoing experimental surgical proce-
dures. Laboratory Animals, 3, 149154.
Suzuki, T., Saito, Y., Isozaki, S., & Ishida, R. (1973). Simple method for portal vein infusion
in the rat. Journal of Pharmaceutical Sciences, 62, 345347.
Thrivikraman, K. V., Huot, R. L., & Plotsky, P. M. (2002). Jugular vein catheterization for
repeated blood sampling in the unrestrained conscious rat. Brain Research. Brain
Research Protocols, 10, 8494.
Tordoff, M. G., Schulkin, J., & Friedman, M. I. (1986). Hepatic contribution to satiation of
salt appetite in rats. The American Journal of Physiology, 251, R10951102.
Torres-Molina, F., Peris, J. E., Garca-Carbonell, M. C., Aristorena, J. C., Granero, L., &
Chesa-Jimnez, J. (1996). Use of rats chronically cannulated in the jugular vein and
the duodenum in pharmacokinetic studies. Effect of ether anesthesia on absorption
of amoxicillin. Arzneimittelforschung, 46, 716719.
Upton, P. K., & Morgan, D. J. (1975). The effect of sampling technique on some blood
parameters in the rat. Laboratory Animals, 9, 8591.
Upton, R. A. (1975). Simple and reliable method for serial sampling of blood from rats.
Journal of Pharmaceutical Sciences, 64, 112114.
Verbaeys, A., Ringoir, S., van Maele, G., & Lameire, N. (1995). Inuence of feeding, blood
sampling method and type of anaesthesia on renal function parameters in the
normal laboratory rat. Urological Research, 22, 377382.
Wan, J., Ernstgrd, L., Song, B. J., & Shoaf, S. E. (2006). Chlorzoxazone metabolism is
increased in fasted SpragueDawley rats. The Journal of Pharmacy and Pharmacology,
58, 5161.
Welberg, L. A., Kinkead, B., Thrivikraman, K., Huerkamp, M. J., Nemeroff, C. B., & Plotsky,
P. M. (2006). Ketaminexylazineacepromazine anesthesia and postoperative
recovery in rats. Journal of the American Association for Laboratory Animal Science,
45, 1320.
Wiersma, J., & Kastelijn, J. (1985). A chronic technique for high frequency blood
sampling/transfusion in the freely behaving rat which does not affect prolactin and
corticosterone secretion. The Journal of Endocrinology, 107, 285292.
Xiang, D. Z., Verbeken, E. K., Lommel, V. A. T., Stas, M., & Wever, D. I. (1998). Composition
and formation of the sleeve enveloping a central venous catheter. Journal of Vascular
Surgery, 28, 260271.
Yang, J., Maarek, J. M., & Holschneider, D. P. (2005). In vivo quantitative assessment of
catheter patency in rats. Laboratory Animals, 39, 259268.
Zeller, W., Weber, H., Panoussis, B., Brge, T., & Bergmann, R. (1998). Renement of
blood sampling from the sublingual vein of rats. Laboratory Animals, 32, 369376.