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Objective To establish a reliable long-term intravenous infusion pathway for freely active rats. Methods Based on the predecessors, the establishment of venous access in free-living rats was improved and beneficially explored.
Establishment and maintenance of venous access in freely moving rats
Summary :
Results 70% of the experimental rats were able to successfully complete the one-month non-continuous infusion experiment, and the operative mortality, infection rate and management loss rate were low. Conclusion The rat intravenous infusion pathway established in this paper is practical and reliable.
Key words : rat; free activity; intravenous infusion pathway medical research activities require various animal models, many models need to establish a long-term infusion venous access in animals, and require infusion or blood collection when animals are awake, free to move [1] ]. The most commonly used animals are rats, as well as monkeys, pigs, rabbits, and mice [2~4]. Clinically, humoral support, intravenous nutrition support, venous blood pressure monitoring, severe trauma, multiple organ dysfunction, and substance addiction in animal experiments require the establishment of venous access [3,5,6]. Based on the predecessors, this paper introduces a reliable and relatively economical technique for establishing and maintaining venous access in rats.
1 animal
Thirty male Sprague 2 Dawley (SD) rats were cleaned at 3 months of age and weighed 250-300 g. They were provided by the Animal Laboratory of Xiangya Second Hospital of Central South University.
2 related equipment and preoperative preparation
2.1 and infusion cannula shaft
The tubing consisted of a Polyurethane Intravascular Tubing BPU-T30 with an inner diameter of 0.58 mm and an outer diameter of 0.91 mm. Polyethylene pipe BPE-T20, inner diameter 0.38 mm, outer diameter 1.09 mm, the polyethylene pipe wall of this specification is thicker and can resist tearing with a certain strength. Infusion tube (COEXTM Tubing, BCOEX-T22) with an inner diameter of 0.61 mm and an outer diameter of 1.60 mm. The inner layer of the infusion tube is polyethylene PE, the resistance is small, the liquid passes through the fluid, the chemical property is stable, and does not chemically react with the infusion component; the outer layer is polyvinyl chloride PVC, which has good tear resistance and durability. A key device in the entire infusion system, the infusion shaft, is to pass the fluid and allow the rat to move freely in the cage. The infusion shaft solves this problem well. It allows the liquid to pass through while it can rotate 360 ​​degrees. The resistance is very small. With a force arm, the rat can easily move in the cage. The infusion shaft used in this experiment was model 375/22, as shown in Figure 1, with a dead volume of 8 μL and a frictional torque of 0.030 oz 2 in oz-in (approximately 2.160 g/cm).
The two ends can be connected to the infusion tube, and the right lower white crossbar is the force arm. The spring tube is connected to the back of the rat. Note that the ends are smooth surfaces, no thread, and the thread should be unclear.
2 . 2 fixing and protection device
To prevent the rat from biting the infusion tube, there must be protection and fixation. The infusion tube from the infusion shaft to the back of the rat passes through a 25cm long spring tube (electric furnace wire). The upper end of the spring tube is connected to the infusion shaft, and the lower end is connected. A plastic piece. The rat infusion puts a vest made of a bonding cloth on its forelimbs. The back of the vest has a plastic piece that can be mated with the plastic part at the lower end of the spring tube and has a fastening device. The infusion tube is from the spring tube through the plastic piece to the subcutaneous vein of the rat to the external jugular vein. The whole infusion tube has no exposed part, so it can effectively prevent the rat from biting. The entire path is shown in Figure 2, but the Harness is replaced by a homemade vest.
2 . 3 preoperative preparation
The BPU tube was cut into 4 cm long sections, the BPE tube was cut into 10 cm lengths, the BPU tube was thickened with chloroform, and one end was placed 1 cm outside the BPE tube to form a 13 cm long tube. Make a knot in the joint to make it "L" shape. Because there is a loop, there is a small circle at the corner of "L". The prepared tube was immersed and disinfected with 75% alcohol, and washed with physiological saline. Soaked in physiological saline before surgery. The 1 mL syringe needle is used as a flathead. Prepare a set of ophthalmic surgical equipment and an epidural needle to facilitate subcutaneous tunneling in the back and neck of the rat.
3 surgical procedures
3.1 Separation vein
The weight of the rats was 250-280 g during operation. The rats were anesthetized with 4% pentobarbital sodium 50 mgPkg, and atropine 013 mg/kg was given at the same time. The skin is placed on the back of the rat's back, and a small opening is made, about 1 to 2 mm long, for the cannula to pass through from the back. The rat was turned over, the head was facing the surgeon, and the right side of the neck was prepared. From the mandible to the upper edge of the clavicle, cut a small mouth, about 2 cm long, separate the subcutaneous tissue, find the right external jugular vein (some experiments can choose femoral vein [7]) and separate, drop the appropriate amount of normal saline, waterproof and dry.
3.2 intubation
Ligation of the distal end of the vein, using a ophthalmic scissors to cut a "V" shape on the vein, do not cut the vein, first insert a small piece of BPE-T10 tube (inner diameter 0.28 mm, outer diameter 0.60 mm) into the vein, because the tube is very thin It is easier to insert. Then use BPE-T10 to make the “V†shape mouth larger. The “L†shaped tube is connected with a 1 mL syringe at the distal end. The syringe needle is flat. The front end of the cannula is cut into a slope, and the tube is filled with physiological saline. Use a pair of tweezers to hold the BPU-T30 and insert a “V†shaped mouth and insert a vein of 3.5 to 4.0 cm.
3 . 3 fixed cannula
Fix the cannula on the vein, because the intubated tube has a 1 cm BPU tube that is placed outside the BPE tube, and the BPE-T20 tube wall is thicker, so the knot on the blood vessel can be slightly forced. Securely secure without worrying that the lumen will be closed. Use a surgical needle to connect the small intubation circle and sew it on the muscle next to the blood vessel. The coil should be slightly loose to prevent muscle necrosis.
3 1 4 tunnels and stitching, wearing vests
Use an epidural needle to puncture the subcutaneous tunnel from the neck incision to the small back of the mouth, remove the 1 mL syringe, insert the cannula from the tunnel through the back of the rat, connect the 1 mL syringe again, and withdraw the blood to confirm the intubation in the blood vessel. Suture the neck incision. 200,000 u penicillin was inserted from the cannula, and the vest was put on the end of the cannula with a plug. Because the rat body was soft during anesthesia, the vest was not too tight to prevent the rat from suffocating. Place the rats in a single cage and keep warm until they wake up.
4 postoperative maintenance and infusion
After the operation, the rats were rested for 3 days, and 200,000 u of penicillin was injected daily from the venous cannula, and then 100,000 u was given daily. At the same time, the drug can be used as a smooth pipe. Liver toxicity can not be used as a routine medication. If the channel pressure is increased, heparin can be temporarily given 200-500 u. Rats were placed in an infusion cage during the experiment. Self-administration experiments were controlled using behavioral cages and corresponding behavioral software. Connect the infusion tube to the syringe pump, and connect the PE tube on the back of the rat to the infusion tube in the spring tube. After the infusion is completed, the rat can be taken out from the infusion cage. The end of the BPE tube is covered with a plastic plug and placed in the cage. support. The infusion is generally shown in Figure 3.
Because the infusion system returns to the cage when the rats are not infused, the air in the pipeline should be drained every time the pipeline is connected, avoid air embolism, and pay attention to aseptic operation.
5 results
Thirty rats died during surgery anesthesia, 2 died the next day after surgery, and the remaining 27 were blocked in 5 of the way. No infection occurred. A total of 22 patients completed the self-administration experiment for 1 month.
6 discussion
Developed countries have been infusing small animals for 40 to 50 years in experimental research [8]. Since the infusion shaft was invented by Michael Loughnane (who later commercialized and established INSTECH SOLOMON) in the 1960s, Numerous commercial companies have developed a number of complementary products for animal infusion, such as a wide variety of catheters, infusion shafts, fixtures, syringe pumps, cages, software, etc. for a variety of animals. The country has a history of nearly 20 years in this respect. A few scientific research units have carried out useful explorations on this technology and designed some inexpensive and practical devices [6, 9~11].
In the experiment, there are several links in the animal infusion. The first is the infusion tube and the infusion shaft, the second is the fixation and connection method of the intubation, and the third is the protection of the pipeline during infusion and the obstacle to reduce the free movement of the rat. Postoperative maintenance. From these aspects, the system reported in China has some problems. For example, the infusion tubing from the syringe pump to the rat vein is entirely made of silicone tubing [12], and the caliber is also different, with an outer diameter of 019 mm, 112 mm, and the like. Silicone tube can effectively reduce the rejection reaction, but the material is soft, such as silicone tube in the whole process. First, it can not be tightened when the vein is inserted. Otherwise, there is danger of closing the tube [12], so the tube is easy to fall off. Second, the tube is infusion. Although there is a spring tube protection outside, there is still the possibility of being stretched and expanded, resulting in inaccurate dose, which is especially important for self-administration experiments, because the self-administration experiment is a non-continuous infusion, each dose is more Small, the system is required to accurately and timely pump the micro-drug into the animal; the third is that the whole pipeline is not interrupted by the silicone tube, and the rats are kept in the infusion cage 24 hours after the operation, until the end of the experiment. Such a rat strap spring tube is easy to inhibit its voluntary activity; it is extremely inconvenient to change the drug in the middle of the experiment; it is difficult to remedy the accidental damage of the pipeline; other experiments cannot be performed without administration, such as behavioral conditional position preference, etc. [3 ]. In this experiment, the inner tube of the rat is connected with the BPE tube by the BPU tube. The BPU rejection reaction is similar to that of the silicone tube, and the BPE tube is firm, thus effectively solving the above problems. Infusion shaft, the most technical part of this infusion system, there is no similar product sold in domestic enterprises. Many infusion systems reported in the literature use similar devices designed by themselves, such as syringe-like structures with movable cannula and bearings, or scalp needles rotating in rubber to achieve 360-degree rotation during infusion [9,13,14] . These designs will inevitably suffer from defects such as leakage, large resistance, and large dead space, which is difficult for long-term experiments. The relationship between the fixation of the cannula in the rat and the intubation has been described before. After the cannula is pierced from the back of the rat, it is fixed with a vest, and the cannula is perpendicular to the back of the rat to avoid rat biting. The vest and the spring tube can be easily docked and separated by using plastic parts. Some systems expand the end of the spring tube into a coil and sew the coil on the back of the rat [10,15], so that the rat is susceptible to infection and pain during activity. The length of the spring tube matches the size of the cage to prevent the spring from being too loose or too tight. The space of the cage should not be too large or too small. The cage size of this experiment is 30 cm × 30 cm × 30 cm. The boom of the infusion fashion infusion shaft can use the principle of leverage to install a suitable load after the fulcrum. Because of the long arm length of the rat, it can effectively reduce or offset the load on the back of the rat. Note that although the infusion shaft can be rotated 360 degrees, the rat can only achieve relative free movement, and the tumble rotation movement is limited. Long-term intravenous administration of rats is a complicated experimental technique. Not only is surgery difficult, but the entire infusion equipment has special requirements. Each subtle link may affect the results of the whole experiment. The method reported in this paper has an obvious The shortcoming is that a large number of foreign related products are used, and the purchase is inconvenient, but it is a wise choice to ensure the smooth progress of the experiment. Only by using the standard experimental device can the accurate and reliable experimental results be easily verified by other laboratories and thus recognized by the peers.