J OLJRNAL
Vol.
OF
38, No.
APPLIED PHYSIC)LOGY 2, Febru ary 1975.
Printed
in
Permane-mt canwlation
R. SABLE-AMPLIS
AND
Institut
31400
de Physiologic,
of the hepatic portal vein in rats
D. ABADIE To&use,
France
SABLE-AMPLB, R., AND ID. ABADXE. Permanent cumulation of the hepatic portal win in rats. J. Appl. Physiol. 38(Z) : 358-359. 1975.-A technique is described for chronic cannulation of the hepatic portal vein in rats. The T-shaped cannula is made in the laboratory from polyethylene tubing. Implantation of the cannula allows injection or blood withdrawal in a small laboratory animal which is unanesthetized and unrestrained. Simultaneous implantation of intracarotid and intraportal cannulas permits comparisons of blood composition in order to study the role of the liver or intestinal absorption. T-shaped withdrawal
cannula;
polyethylene
tubing;
drug
injection;
(4 mg/ml) saline, and the long end closed with a suitably large nylon pin made from fishing leader. Imfdan tat ion. The animal is anesthetized with pentobarbital incision is made and the duodenum (25 mg/kg, ip>. A ventral very carefully reflected to the animal’s left to expose the portal vein. The vein is freed from connective tissue for a distance of about 0.5 cm between the two pancreatic-duodenal veins (Fig. 1). Loose ligatures are passed under the vein at each end of the cleared area and the circulation is interrupted with hemostats. After hemisection of the vein, one end of the short portion of the cannula is inserted and tied in place. The vessel is then completely sectioned and the other end of the short piece inserted and tied. Circulation is reestablished after opening the free (long portion) end of the cannula to prevent passage of air emboli or blood clots into the general circulation. The free end is then filled with
blood
EXPERIMENTS on the chronic effects of various drugs required a comparison of blood from the general circulation with that from the hepatic portal vein. To study drug effects in unanesthetized and unrestrained rats, we have developed methods to place T-shaped cannulas in the common carotid artery and in the hepatic portal vein. The cannulation technique for the carotid has already been described in rats and in rabbits (2). The technique for implanting a T-shaped cannula in the hepatic portal vein of rats is given here. Also given are changes in glucose and insulin composition of carotid and portal blood in the same animal during 3 h following glucose loading.
MATERIALS
AND
/L
Cannula
Lwe
Pancrea
duodena
METHODS
icve ins
-Duodenum
Preparation of ca nnula. The T-shaped cannula is made in the laboratory with Clay-Adams polyethylene tubing. The short portion of the cannula (equivalent to the upper cross bar of the letter T) consists of a M-cm length of PE-205 (ID 0.062 in x OD 0.082 in) for a 250-g rat. This permits lengthwise insertion into the portion of the portal vein to be cannulated. The long portion of the cannula (equivalent to the vertical bar of the letter T) consists of a 12- to 15-cm length of PE-60 (ID 0.030 in x OD 0.048 in) or PE-90 (ID 0.024 in x OD 0.050 in). This length was found suitable for exteriorization at the back of the animal to permit blood collection. To join the two pieces of tubing, the short piece is pierced in the middle with a hot metal needle of suitable diameter. A metal wire mandrel is inserted through the long piece and into the hole made in the short piece. This serves as a support when the walls of the long piece brought into contact with the edges of the hole. The joint is then sealed with heatliquefied polyethylene tubing. This is done by partially melting (until a small ball of liquid is formed) the tip of a length of PE-20. The two ends of the short portion are very gently flared with a match. This procedure smooths out any ridges which might enhance blood clotting and also prevents slipping of the ligatures. Cannulas are prepared 1 or 2 days before use and placed in a germicide solution. The cannulas are also siliconized by placing them several hours before in a 2% solution of “Emulsion Si 35 B Rhbne Poulenc,” followed by drying in an oven at 70°C. Just prior to use, they are rinsed and filled with heparinized
FIG.
1. Drawing
showing
shape
of cannula
and site of implanta-
tion, heparinized saline and heat-seared. The heat-sealed end of the cannula is tied to a large needle, passed under the skin, and exteriorized at the back of the neck. Immediately following the operation which is done as aseptically as possible, the wound is powdered with sulfonamide and the animal injected with 20,000 units of penicillin. As soon as the animal is recovered from the anesthetic, the cannuta is flushed, refilled with heparinized saline, and then cut to the desired length. Normally 2-3 cm of tubing is left exposed. Closure is with a nylon pin. The use of a nylon pin as opposed to continuously cutting and heat sealing avoids the necessity of leaving a long piece of tubing outside of the animal. Cannulas remain functional for varying periods of time. If sufficient precautions are observed during the surgery, if the cannula is carefully made (no intrusion of the long piece into the lumen of the short piece), and if daily flushings are performed, patency can be extended. For example, of every 10 animals, 8 were found suitable for use at the end of 1 wk while 5 of every 10 animals were suitable for use at 1 mo following the surgery. With regard to survivaI, if an animal survived until compIetion of the surgery, 358
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 10, 2019.
CANNULATION
OF
PORTAL
VEIN
IN
RAT
359
insulin
b30
‘v-
!
-_
---_
d ca rot 80?.
glucose lnsul
_ In
Id
--Porta
---_
-V
DISCUSSION
1 vem
w-m
A_. -. -. .A
v---JJ
0 m. . . ..*o
hours
FIG.
hepatic
2. Changes in glucose and portal vein plasma following
Blood is allowed to drop freely into a receptacle. After blood withdrawal the cannula is filled with a calculated (from length and diameter) volume of heparinized saline and closed with the nylon pin. Illustration. As an example of the usefulness of the technique, effects of a glucose load on plasma glucose levels were determined. Results are presented in Fig. 2. Six rats provided with intracarotid and portal cannulas 8 days before the experiment were given a perfusion of a 30y0 glucose solution into carotid artery (1.5 g/kg over a 5-min period). Blood samples were taken simultaneously from the carotid and portal cannulas before perfusion, and then 15 min, 1 h, and 3 h after perfusion. Plasma glucose was measured by the method of Nelson-Somogyi (1) and insulin (IRI) by radioimmunoassay (3). Results of a representative experiment are given in Fig. 2.
insulin levels in glucose loading
carotid and (1.5 g/kg).
mortality rate was zero. If the cannulas became obstructed, collateral circulation developed rapidly. Rats are caged individually; their health and activity have posed no problems. Blood sa@ing. To draw blood, the nylon pin which serves as a stopper is removed and the contents of the cannula discarded.
The example given here demonstrates the advantages of simultaneous implantation of intracarotid and intraportal cannulas in the same animal. The technique described here lends itself to numerous other applications, as much for liver as for intestinal biochemistry and physiology. It allows simultaneous injection and easy blood withdrawal in an unanesthetized and unrestrained animal. We acknowledge with thanks Mrs. M. Forastiero for help in the development of this technique. This study was supported by a grant from Mr, L. Lareng, President of the University Paul Sabatier, Toulouse. Received
for publication
I7 May
1974.
REFERENCES 1. NELSON, N. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153 : 375-380, 1944. 2. SABLE-AMPLTS, R., C. CAYROL, AND R. AGTD. Canulation en T
de la carotide et de la jugulaire chez le rat et le lapin. Applications pharmacologiques. J. Pharmacol., Paris 4 : 363-367, 1973. 3. YALOW, R. S., AND S. A. PERSON. Immunoassay of endogenous plasma insulin in man. J. C&n. I~est, 39 : 1157-l 175, 1960.
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 10, 2019.
Vol.
OF
38, No.
APPLIED PHYSIC)LOGY 2, Febru ary 1975.
Printed
in
Permane-mt canwlation
R. SABLE-AMPLIS
AND
Institut
31400
de Physiologic,
of the hepatic portal vein in rats
D. ABADIE To&use,
France
SABLE-AMPLB, R., AND ID. ABADXE. Permanent cumulation of the hepatic portal win in rats. J. Appl. Physiol. 38(Z) : 358-359. 1975.-A technique is described for chronic cannulation of the hepatic portal vein in rats. The T-shaped cannula is made in the laboratory from polyethylene tubing. Implantation of the cannula allows injection or blood withdrawal in a small laboratory animal which is unanesthetized and unrestrained. Simultaneous implantation of intracarotid and intraportal cannulas permits comparisons of blood composition in order to study the role of the liver or intestinal absorption. T-shaped withdrawal
cannula;
polyethylene
tubing;
drug
injection;
(4 mg/ml) saline, and the long end closed with a suitably large nylon pin made from fishing leader. Imfdan tat ion. The animal is anesthetized with pentobarbital incision is made and the duodenum (25 mg/kg, ip>. A ventral very carefully reflected to the animal’s left to expose the portal vein. The vein is freed from connective tissue for a distance of about 0.5 cm between the two pancreatic-duodenal veins (Fig. 1). Loose ligatures are passed under the vein at each end of the cleared area and the circulation is interrupted with hemostats. After hemisection of the vein, one end of the short portion of the cannula is inserted and tied in place. The vessel is then completely sectioned and the other end of the short piece inserted and tied. Circulation is reestablished after opening the free (long portion) end of the cannula to prevent passage of air emboli or blood clots into the general circulation. The free end is then filled with
blood
EXPERIMENTS on the chronic effects of various drugs required a comparison of blood from the general circulation with that from the hepatic portal vein. To study drug effects in unanesthetized and unrestrained rats, we have developed methods to place T-shaped cannulas in the common carotid artery and in the hepatic portal vein. The cannulation technique for the carotid has already been described in rats and in rabbits (2). The technique for implanting a T-shaped cannula in the hepatic portal vein of rats is given here. Also given are changes in glucose and insulin composition of carotid and portal blood in the same animal during 3 h following glucose loading.
MATERIALS
AND
/L
Cannula
Lwe
Pancrea
duodena
METHODS
icve ins
-Duodenum
Preparation of ca nnula. The T-shaped cannula is made in the laboratory with Clay-Adams polyethylene tubing. The short portion of the cannula (equivalent to the upper cross bar of the letter T) consists of a M-cm length of PE-205 (ID 0.062 in x OD 0.082 in) for a 250-g rat. This permits lengthwise insertion into the portion of the portal vein to be cannulated. The long portion of the cannula (equivalent to the vertical bar of the letter T) consists of a 12- to 15-cm length of PE-60 (ID 0.030 in x OD 0.048 in) or PE-90 (ID 0.024 in x OD 0.050 in). This length was found suitable for exteriorization at the back of the animal to permit blood collection. To join the two pieces of tubing, the short piece is pierced in the middle with a hot metal needle of suitable diameter. A metal wire mandrel is inserted through the long piece and into the hole made in the short piece. This serves as a support when the walls of the long piece brought into contact with the edges of the hole. The joint is then sealed with heatliquefied polyethylene tubing. This is done by partially melting (until a small ball of liquid is formed) the tip of a length of PE-20. The two ends of the short portion are very gently flared with a match. This procedure smooths out any ridges which might enhance blood clotting and also prevents slipping of the ligatures. Cannulas are prepared 1 or 2 days before use and placed in a germicide solution. The cannulas are also siliconized by placing them several hours before in a 2% solution of “Emulsion Si 35 B Rhbne Poulenc,” followed by drying in an oven at 70°C. Just prior to use, they are rinsed and filled with heparinized
FIG.
1. Drawing
showing
shape
of cannula
and site of implanta-
tion, heparinized saline and heat-seared. The heat-sealed end of the cannula is tied to a large needle, passed under the skin, and exteriorized at the back of the neck. Immediately following the operation which is done as aseptically as possible, the wound is powdered with sulfonamide and the animal injected with 20,000 units of penicillin. As soon as the animal is recovered from the anesthetic, the cannuta is flushed, refilled with heparinized saline, and then cut to the desired length. Normally 2-3 cm of tubing is left exposed. Closure is with a nylon pin. The use of a nylon pin as opposed to continuously cutting and heat sealing avoids the necessity of leaving a long piece of tubing outside of the animal. Cannulas remain functional for varying periods of time. If sufficient precautions are observed during the surgery, if the cannula is carefully made (no intrusion of the long piece into the lumen of the short piece), and if daily flushings are performed, patency can be extended. For example, of every 10 animals, 8 were found suitable for use at the end of 1 wk while 5 of every 10 animals were suitable for use at 1 mo following the surgery. With regard to survivaI, if an animal survived until compIetion of the surgery, 358
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 10, 2019.
CANNULATION
OF
PORTAL
VEIN
IN
RAT
359
insulin
b30
‘v-
!
-_
---_
d ca rot 80?.
glucose lnsul
_ In
Id
--Porta
---_
-V
DISCUSSION
1 vem
w-m
A_. -. -. .A
v---JJ
0 m. . . ..*o
hours
FIG.
hepatic
2. Changes in glucose and portal vein plasma following
Blood is allowed to drop freely into a receptacle. After blood withdrawal the cannula is filled with a calculated (from length and diameter) volume of heparinized saline and closed with the nylon pin. Illustration. As an example of the usefulness of the technique, effects of a glucose load on plasma glucose levels were determined. Results are presented in Fig. 2. Six rats provided with intracarotid and portal cannulas 8 days before the experiment were given a perfusion of a 30y0 glucose solution into carotid artery (1.5 g/kg over a 5-min period). Blood samples were taken simultaneously from the carotid and portal cannulas before perfusion, and then 15 min, 1 h, and 3 h after perfusion. Plasma glucose was measured by the method of Nelson-Somogyi (1) and insulin (IRI) by radioimmunoassay (3). Results of a representative experiment are given in Fig. 2.
insulin levels in glucose loading
carotid and (1.5 g/kg).
mortality rate was zero. If the cannulas became obstructed, collateral circulation developed rapidly. Rats are caged individually; their health and activity have posed no problems. Blood sa@ing. To draw blood, the nylon pin which serves as a stopper is removed and the contents of the cannula discarded.
The example given here demonstrates the advantages of simultaneous implantation of intracarotid and intraportal cannulas in the same animal. The technique described here lends itself to numerous other applications, as much for liver as for intestinal biochemistry and physiology. It allows simultaneous injection and easy blood withdrawal in an unanesthetized and unrestrained animal. We acknowledge with thanks Mrs. M. Forastiero for help in the development of this technique. This study was supported by a grant from Mr, L. Lareng, President of the University Paul Sabatier, Toulouse. Received
for publication
I7 May
1974.
REFERENCES 1. NELSON, N. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153 : 375-380, 1944. 2. SABLE-AMPLTS, R., C. CAYROL, AND R. AGTD. Canulation en T
de la carotide et de la jugulaire chez le rat et le lapin. Applications pharmacologiques. J. Pharmacol., Paris 4 : 363-367, 1973. 3. YALOW, R. S., AND S. A. PERSON. Immunoassay of endogenous plasma insulin in man. J. C&n. I~est, 39 : 1157-l 175, 1960.
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