Surgical techniques and first aid experiments on laboratory animals
Surgical techniques and first aid experiments on laboratory animals
The experimental method was obtained from the official website of the Fourth Military Medical University
Operation method
Surgical techniques and first aid experiments on laboratory animals Move I. Basic surgical techniques commonly used in laboratory animals Figure 1 2. Hemostasis Hemostasis is an important part of surgical operation. Whether hemostasis is perfect or not during surgery not only directly affects the exposure of the surgical site and surgical operation, but also relates to the safety of the animals after surgery, the healing of the incision and whether it causes complications. Intraoperative hemostasis must be accurate, rapid and reliable. Commonly used methods of hemostasis are: 3. Tissue separation method The purpose of separating tissues is to fully reveal the deep tissues, resulting in surgical pathways to facilitate the resection of the lesion. Operation should pay attention to the following principles: ① in the same plane and strive to make a vertical incision to ensure that the edge of the incision is neat, prohibit oblique cuts and saw cuts, in order to reduce the damage and facilitate healing. ② when cutting multiple layers of tissue, generally should be cut according to the tissue level, the incision size should be appropriate. ③ When cutting the muscle, it should generally be done in the direction of the muscle fibers. ④ It is important to ensure the smooth drainage of secretions from incision trauma. There are two methods of tissue separation: Figure 6 Figure 7 Figure 8 (2) Femoral arterial and venous cannulation: In acute experiments on large animals, femoral arterial and venous cannulation is often required to facilitate the administration of drugs through arteries and veins at any time. Take femoral vein cannulation in rabbits as an example. Separate a section of the femoral vein with mosquito hemostatic forceps, wear two lines underneath it, one first ligated at the distal end of the vein, and then gently lift the anterior wall of the vein tube near the central end of the forceps, and then use ophthalmic scissors to straighten the cuts, at an angle of 45 °, and cut the diameter of the blood vessel of 1/3, pay attention to the blood vessel incision surface must be an inverted "V"-like diagonal cut and then inserted a catheter, and then use the other line to insert a catheter into the femoral vein. The venous catheter is inserted and secured in the lumen of the vein with another wire. For more product details, please visit Aladdin Scientific website.
(I) Basic surgical techniques
1. incision According to the experimental requirements to determine the location and size of the surgical incision. When cutting the skin first taut, the blade of the knife and the skin perpendicular to the force to be appropriate, a cut the whole layer of the skin, cut the seam neatly without skewing. When cutting the skin and subcutaneous tissues, it is generally required to cut layer by layer according to the anatomical level, pay attention to hemostasis, and avoid damaging the important tissues and organs in the deeper layers (Fig. 1, Fig. 2).
Figure 2
(1) Preventive hemostasis: use some drugs that can improve blood coagulability within 1~2h before surgery to reduce intraoperative bleeding. Commonly used prophylactic hemostatic agents are 10% calcium chloride solution, 10% sodium chloride solution.
When local anesthesia, with the application of epinephrine, that is, adding 0.1% epinephrine 2 ml in 1000 ml of procaine solution, using its vasoconstriction effect, to achieve the purpose of reducing the bleeding of the surgical site. In the end of the limbs, penis, tail surgery, in order to avoid excessive bleeding, the tourniquet can be wrapped around the top of the surgical site, and then loosened after the surgical site is completely hemostatic.
(2) intraoperative hemostasis: ① pressure hemostasis: bleeding during surgery can generally be first sterilized gauze or wrung out warm saline gauze pressure for a few moments, do not use dry gauze to wipe, in order to reduce tissue damage. ② clamping hemostasis: use hemostatic forceps and blood flow direction perpendicular to the severed end of the blood vessel, stay for a period of time after removing the hemostatic forceps. ③ Ligation hemostasis: commonly used for bleeding from ineffective compression or larger vessels. Bleeding point with gauze compression dipping suction, with hemostatic forceps clamp the severed end of the blood vessel one by one, to clamp accurate, clamping, but should try to clip the surrounding tissues, and then ligature hemostasis with silk thread. Ligation, the first vertical hemostatic forceps, the ligature line around the pliers under the clamping point, and then flatten the pliers after the tip of the pliers slightly cocked, playing the first knot, the edge of the tightening side of the gently release the hemostatic forceps, and then play the second knot. The types of knots are the square knot (flat knot), the surgical knot and the triple square knot (Figure 3). There are two methods of tying knots: hand-tying and instrument-tying (Figures 4 and 5). （There are two methods of knotting: hand knotting and instrument knotting (Figure 4, Figure 5). (4) Burning to stop bleeding: burn the broken ends of blood vessels with a hot iron to make the blood and tissues coagulate, so as to achieve the purpose of stopping bleeding. ⑤ Pharmacological hemostasis: when the internal organs bleed, use gauze to suction the accumulated blood, and then apply hemostatic powder, Yunnan Baiyao or thrombin on the wound surface, and apply slight pressure for 5-10s to stop bleeding.
(1) Sharp separation method: using sharp instruments such as knives and scissors for direct cutting, this method is used for fine dissection of skin, mucous membranes, various tissues and separation of tight adhesions.
(2) Blunt separation method: the use of knife handles, hemostats, strippers or fingers, etc. to separate the loose connective tissues of the muscle and fascial interstices.
Soft tissue separation requirements according to the anatomical level of separation layer by layer, to maintain a clean and clear field of vision. In principle, the main blunt separation, if necessary, can also use the knife, scissors. (1) Separation of connective tissue: insert vascular forceps to open it up and make blunt separation. For the thin layer of fascia, a knife and scissors can be used when the absence of blood vessels is confirmed. For thick layers of fascia, as they often contain blood vessels and are not easily visualized, scissors should not be used easily. When using vascular forceps for blunt dissection, it should be slowly layered, from shallow to deep, avoiding blood vessels. If you need to use a sharp instrument, you should use two vascular forceps for double clamping (sometimes even ligation), and then cut between the two clamps. ② separation of muscle tissue: should be in the whole muscle and other tissues, a piece of muscle and another piece of muscle demarcation, along the direction of the muscle fibers for blunt separation, muscle tissue containing small blood vessels, if you need to cut off, should be made beforehand with vascular forceps for double clamping, ligature before you can cut. (iii) Separation of vascular nerves: Follow the direction of its straight line, carefully separate it with a glass minute needle, and do not pull it laterally.
4. Suture methods There are many suture methods, but they are summarized as interrupted suture, continuous suture, and blanket suture (Figure 6), as well as tension-reducing suture, decubitus suture, purse-string suture, and "8" suture. Interrupted suture is the most commonly used suture, general tissue can be used; continuous suture is commonly used to suture the peritoneum and gastrointestinal tract, etc., faster, and has a certain hemostatic effect; blanket edge suture is commonly used for skin grafting suture, gastrointestinal anastomosis suture posterior wall, etc., the edge of the neatly, and have a certain effect of hemostasis; mattress suture is commonly used in the gastrointestinal tract, vascular sutures, etc., the tension-reduced suture is commonly used for suture of the skin, and can be combined with other sutures. Decompression suture is commonly used to suture the skin, and can be used in conjunction with other sutures, its characteristic is that the entry and exit holes of the suture are farther away from the edge of the trauma (2-4cm), or a gauze pillow is installed before tying the knot in order to reduce the tension of the tissues and prevent the tissues from being torn by the suture; the purse-string suture is commonly used to suture small perforation in the gastrointestinal tract and embedding of appendiceal stumps, etc.; the "figure of 8" shape is commonly used for suturing fascia, tendon membranes and muscles.
Before suturing, hemostasis should be performed thoroughly, and the cavity should be cleared of foreign bodies, clots and necrotic tissue. The entry and exit holes of the suture needle should be symmetrical and about 0.5 to 1 cm from the traumatic edge. The sutures should be appropriately tightened and loosened. The knot should be concentrated on the same side of the wound edge. If necessary, consider making a tension-reducing suture and leaving a drainage hole. When suturing, the aseptic routine must be observed.
External wound sutures need to be removed after a certain period of time (about 7-14d). Depending on the condition of the wound suture, it may be decided to remove it in stages or at once. In case of septic wounds, all or part of the sutures are removed according to the therapeutic needs. Before suture removal, the suture area, especially on the sutures and needle holes, should be sterilized with iodine and alcohol. When removing sutures, attention needs to be paid to the method of suture removal (Figure 7)
1. Neck surgery
(1) Tracheal intubation: take rabbits as an example. Commonly used surgical instruments, silk threads, cotton balls, "Y"-shaped tracheal tube, surgical table, 20% urethane, etc. are required for the operation. Before surgery, the animals were anesthetized (20% ulatan 1g/kg body weight by intravenous injection at the ear margin), fixed in the supine position, and the skin was prepared. Then, a scalpel was used to make a skin incision of about 5-7 cm in length in the neck, from the lower edge of the thyroid cartilage, along the lower midline, to expose the sternohyoid muscles. A vascular forceps was inserted between the right and left sternocleidomastoid muscles for blunt separation (two index fingers could also be used for separation). When separating, the vascular forceps should not be inserted too deeply to avoid damaging small blood vessels. The two muscles were pulled apart to both sides, exposing the trachea for about 5 cm. the trachea was separated from the connective tissue behind it with curved vascular forceps, and threaded for spare. A scalpel or surgical scissors are used to make an inverted "T"-shaped incision between the two cartilaginous rings of the trachea 1 cm below the thyroid cartilage. The incision should not be larger than 1/3 of the diameter of the trachea, and if there is blood or secretion in the trachea, use a small cotton ball to wipe it out first to ensure a smooth airway. Then, hold the corner of the incision with forceps and insert a tracheal tube of appropriate caliber into the trachea from the incision to the chest end, tie it firmly with a spare wire and fix it on the side tube to avoid dislodging.
(2) Separation and intubation of cervical blood vessels: firstly, peel off the nerves and blood vessels, insert a hemostat of appropriate size into the connective tissue near the nerves or blood vessels, expand the hemostat in the direction of the nerves and blood vessels, and gradually peel off the connective tissue around them. When separating small nerves or blood vessels, first use mosquito hemostatic forceps or glass parting needles to separate the layers of connective tissue around the nerves or blood vessels along the direction of nerve or blood vessel travel, and then use glass parting needles to separate the nerves or blood vessels intact. Finally, an ophthalmic forceps is used to pass a thin thread soaked in saline underneath the nerve or blood vessel for lifting and ligating the nerve or blood vessel. After the separation is complete, a piece of gauze soaked in warm saline is placed over the incision tissue to keep the incision site moist.
1) External jugular vein-right atrium cannulation: In acute experiments, external jugular vein cannulation is commonly used for injecting various drugs, taking blood, transfusing fluids and measuring central venous pressure. The external jugular vein is thicker in rabbits and dogs and is the main vein trunk of the head and neck. The external jugular vein is very superficial in distribution and is located on the outer edge of the sternocleidomastoid muscle under the skin of the neck. When separating it, the skin is cut on one side, and the connective tissue around the vein is gently separated from it by lifting up the outside of the neck skin with a finger to see the dark purplish-red external jugular vein, and using blunt-ended hemostatic forceps or a glass parting needle in the direction of vascular travel.
Before cannulation of the external jugular vein, a plastic or silicone tube of appropriate length and inner diameter of 0.1-0.2 cm is prepared, with the insertion end cut into a beveled surface and the other end connected to an infusion or venous pressure measurement device. The proximal end of the vein is first clamped with an arterial clip during cannulation, and then the distal end is ligated after the vein fills. Using ophthalmic scissors, a small horseshoe-shaped cut is made at a 45° angle on the vein against the distal ligature, about one-third or one-half the diameter of the tube, and the catheter is inserted. Tie a knot in the spare line, remove the arterial clip, and slowly feed the catheter toward the right atrium to the desired length. When measuring central venous pressure, rabbits need to be inserted about 5cm, dogs inserted about 15cm, at this time the catheter mouth in the superior vena cava vein near the entrance of the right atrium, can be observed from the central venous pressure detector that the fluid level stops dropping and fluctuates markedly with respiration, ligature to fix the catheter. If the external jugular vein is used for injection, infusion, etc., the catheter is usually sent in 2-3cm.
(2) Separation and cannulation of the common carotid artery: In acute experiments, the common carotid artery catheter was used for measuring arterial blood pressure or bloodletting. The common carotid artery is located on the lateral side of the trachea, and its ventral surface is covered by the sternocleidomastoid muscle and sternocleidomastoid muscle. When the connective tissue between the two muscles is separated, a pinkish-red, thicker blood vessel can be found, which is called the common carotid artery when touched with a finger and felt as pulsation. The common carotid artery and the cervical nerves are bound together by a connective tissue membrane called the cervical vascular nerve bundle. The left thumb and forefinger grasp the cervical skin and cervical muscle, and the middle finger is used to lift up the ectopia, and the right hand holds a mosquito hemostat or a glass split needle to separate the common carotid artery in the direction of the vascular nerve course. During the operation, attention should be paid to the common carotid artery in the vicinity of the thyroid gland has a large side branch, for the anterior thyroid artery, do not cut it off when separating. During isolation, the surgical field should be frequently moistened with saline and swabbed of nearby blood. In order to facilitate intubation or operations such as the common carotid artery pressor reflex, the common carotid artery should be separated as long as possible (2-3 cm in rats and guinea pigs, 3-4 cm in rabbits, and 4-5 cm in dogs).
The catheter used for common carotid artery cannulation is the same as the external jugular vein catheter, which is filled with heparin saline solution. Two spare wires were placed under the separated common carotid artery; one wire was used to ligate the distal end of the artery, the proximal end was clamped with an arterial clip, and the other wire was tied with a live knot between the arterial clip and the distal end of the ligated wire. The vascular incision is the same as that of the external jugular vein. The catheter was inserted into the lumen of the artery for 1 to 2 cm, and then a knot was tied with a wire, which was tightened to the extent that it would not bleed after releasing the arterial clip. The ligature was fixed and then knotted around the catheter to avoid catheter slippage. Do not release the arterial clip before measurement.
(3) Distribution and separation of the vagus, sympathetic and decompression nerves in the neck.
(1) Distribution of vagus, sympathetic and decompression nerves in the neck. (1) Rabbit: After the trachea is isolated in the neck, its lateral side is formed by the connective tissue of the encircling common carotid artery with three nerves of different thicknesses to form a vascular nerve bundle. The thickest one is the vagus nerve, which is white; the thinner one is the sympathetic trunk of the neck, which is grayish-white, and the sympathetic trunk has branches to the heart; the thinnest one is the decompression nerve, which belongs to the afferent nerve. Its nerve endings are distributed within the walls of the aortic arch. The decompression nerve is generally between the vagus and the sympathetic nerve, but its location often varies, and the rate of variation is very large. Cat: the sympathetic and vagus nerves run side by side, the sympathetic nerve is thinner while the vagus nerve is thicker, and the decompression nerve moves into the vagus nerve. (iii) Dog: only a thick nerve trunk, called the vagus sympathetic trunk, is seen dorsal to the common carotid artery. The junctional ganglion of the vagus nerve is adjacent to the anterior cervical ganglion of the sympathetic nerve. The vagus nerve enters the neck below the first cervical vertebra and travels in close proximity to the sympathetic trunk and is encompassed by a common sheath, uniting to form the vagus sympathetic trunk.
(2) Method of isolation of the vagus, sympathetic, and decompression nerves in the neck. The method of isolation and exposure is the same as for the common carotid artery. The nerves can be identified according to their morphology, location and direction of travel. Ophthalmic forceps can be used to identify the connective tissue membrane near the cervical vascular nerve bundles, gently pulled to the outside, or in the common carotid artery under the thread, gently lifted up, you can see the blood vessels, nerves from top to bottom arranged in the connective tissue membrane. The vagus and sympathetic nerves are easily recognized, while the decompression nerve is only a separate nerve in rabbits, which is easier to recognize, while in humans, horses, pigs, dogs and other animals, this nerve does not travel alone, but walks in the vagus sympathetic trunk or vagus nerve. Because the decompression nerve is thin and easily damaged, it should be separated from the surrounding tissue with a glass parting needle before separating the other nerves, and the length of the separation is generally 2~3cm long. After separation, a fine line moistened with saline should be placed under each nerve, and each nerve should be tied with an imaginary knot and prepared for use.
(3) Separation method of cervical phrenic nerve: cut the skin of the neck, separate the subcutaneous tissues, expose the trachea and sternocleidomastoid muscle, a vein can be seen close to the subcutaneous walking in the outer edge of the sternocleidomastoid muscle, which is the external jugular vein. The external jugular vein and sternocleidomastoid muscle were gently separated to the depth with hemostatic forceps, and when separated to the edge of the trachea, the thicker brachial plexus nerves were seen traveling along the posterior exterior, and there was a thinner nerve on the medial side of it, which crossed the brachial plexus and crossed with it at the lower 1/5 of the neck, and traveled inward and posteriorly, i.e., it was phrenic nerve. After identifying the phrenic nerve, the phrenic nerve was carefully divided into 1~2 cm with glass parting needles and a thread was set up underneath the nerve for spare use. If you need to record the potential in the experiment, you can carefully peel off the connective tissue membrane around the nerve trunk, then you can increase the amplitude of the recorded potential.
2. Thoracic surgery in rabbits as an example. Because of the anatomical characteristics of rabbits: the left and right thoracic cavities are not accessible, and the heart is in the mediastinum outside the pleural cavities on both sides. Therefore, many heart experiments are carried out using this feature, and a method of opening the chest with less bleeding and less likely to break the pleura is now introduced.
The rabbit was fixed in the supine position, the skin was prepared, local anesthesia was applied to the sternal pedicle in the middle of the chest, and a 5-6 cm skin incision was made from 1 cm above the sternal pedicle fossa to the middle of the sternal pedicle. Then the cervical vastus muscle above the sternal fossa was separated, the separation should not be too deep, so as not to damage the vein, the direction of separation was close to the lower sternal stalk, and it was cut with coarse scissors along the middle of the sternal stalk, and it was easy to cut through the pleura if it deviated from the middle. Finally, the heart is fully exposed by traction expansion with a coarse cotton thread through the cut sternal pedicle.
When using scissors, attention should be paid to the upward pick up, in order to prevent cutting the pleura; surgery should minimize bleeding, such as bleeding more, first use hemostatic forceps to clip the bleeding point, and then use the thread ligation to stop bleeding.
3. Abdominal surgery ureteral intubation in rabbits as an example. The operation requires the use of commonly used surgical instruments, silk thread, cotton balls, glass split needles, "Y"-shaped catheter (can be inserted into both sides of the ureter at the same time), rabbit operating table, 20% urethane, etc. Before the operation, the animal was anesthetized and placed on its back. Before surgery, the animal was anesthetized, fixed in supine position, and part of the hair on the upper abdomen of the pubic symphysis was clipped. Then, a median incision was made in the lower abdomen upward from the upper edge of the pubic symphysis, 3-4 cm long, and the abdominal wall and peritoneum were cut along the white line of the abdomen with surgical scissors, taking care not to injure the abdominal visceral organs. The bladder is searched for and turned out of the abdomen, and the ureters are found on both sides of the base of the bladder. Note that the operative field is covered with warm saline gauze to maintain intra-abdominal temperature and humidity. After locating the ureter, a loose knot is fastened with a silk thread near the bladder, and the ureter is lifted up with curved forceps or the little finger, and a small diagonal cut is made with ophthalmologic scissors (at an angle of 45°, about 1/2 of the diameter of the ureter) to insert a catheter (filled with saline beforehand) from the small opening towards the kidney, and secured with a spare silk thread to prevent it from slipping out. After insertion, urine can be seen slowly flowing out of the tube. The catheter should be inserted gently to prevent bleeding.
4. Femoral surgery
(1) Separation of femoral artery, vein and femoral nerve: the blood vessels and nerves in the femoral triangle include femoral artery, femoral vein and femoral nerve, which are under a layer of deep fascia. When separating, mosquito hemostatic forceps can be used at the intersection of pubococcygeus muscle and suture muscle to carefully separate along the medial edge of the posterior part of the suture muscle and gently pull the posterior part of the suture muscle outward, and the vascular and nerve bundles surrounded by the deep fascia can be seen underneath it. After careful separation of the deep fascia, the blood vessels and nerves were completely exposed. The femoral vein was located medially, the femoral nerve was located laterally, and the femoral artery was located in the center (Figure 8). The connective tissue membrane between the vessels and nerves was carefully separated with a glass split-needle or mosquito hemostatic forceps and threaded for backup.
First aid measures for experimental animals
When the experiment is carried out due to anesthesia overdose, large blood loss, excessive trauma, asphyxia and other reasons, and the animal's heartbeat is weakened or even stopped, the blood pressure drops sharply or even can not be measured; respiration is very slow and irregular or even respiration stops, the corneal reflex disappears and other clinical symptoms of death, first aid should be carried out immediately. The method of first aid can be determined according to the animal's condition. The following first-aid measures are commonly used for dogs, rabbits and cats.
1. Artificial respiration: Press the animal's chest with both hands for artificial respiration. If there is an electric artificial respirator, it is feasible to connect the artificial respirator for artificial respiration after tracheal intubation. Once the animal resumes spontaneous breathing, artificial respiration can be stopped.
When the artificial respirator is used, its capacity should be adjusted: 50/min for rats, 8 ml/kg each time, i.e. 400 ml/(kg-min); 30/min for rabbits and cats, 10 ml/kg each time, i.e. 300 ml/(kg-min); 20/min for dogs, 100 ml/kg each time (i.e. 2000 ml/(kg-min).
2. Acupuncture Acupuncture Renzhong acupoint is more effective in rescuing rabbits. It is more effective to stimulate the phrenic nerve with pulse of several hundred times per minute frequency for dogs.
3. Injecting cardiotonic agent can be injected intravenously with 1ml of 0.1% epinephrine, or directly into the heart if necessary. Epinephrine has the effect of enhancing myocardial contractility, increasing myocardial contraction amplitude and accelerating atrial conduction velocity, expanding coronary arteries, enhancing myocardial blood supply, oxygenation and improving myocardial metabolism, and stimulating high and low cardiac pacing points.
When animals injected with adrenaline, such as the heart has been beating but extremely weak, can be injected from the vein or intracardiac cavity with 5% calcium chloride 5 ml. Calcium ions can stimulate myocardial tension, which can make the myocardial contraction strengthened and the blood pressure rise.
4. Injecting respiratory center excitatory drugs
(1) 25% Nicosamide 1ml can be injected intravenously from the animal, which can directly excite the respiratory center of medulla oblongata and accelerate and deepen the respiration; the excitatory effect on the vasomotor center is weaker. The effect is more obvious in the case of animal inhibition.
(2) 1% theophylline 0.5 ml can be injected intravenously from animals, which can stimulate the chemoreceptors of carotid artery body, reflexively excites the respiratory center; at the same time, this drug also has slight direct excitation effect on the respiratory center. As a respiratory excitatory drug, it is more rapid and significant than other drugs. The respiration can be deepened and accelerated rapidly, and the blood pressure also rises at the same time.
5. Arterial rapid injection of hypertonic glucose solution is usually used through the animal femoral artery counterpressure, rapid, shock injection of 40% glucose solution. The amount of injection depends on the animal, such as dogs can be calculated according to 2 ~ 3ml/kg body weight. This stimulates the animal's intravascular receptors and reflexively causes an improvement in blood pressure and respiration.