耳聋动物模型 z-bo 2020-10-15 268

　　The species difference between miniature pigs and humans is very small. They are relatively close to humans in terms of organ structure and metabolism. Many diseases share common causes with humans. It is an ideal animal model for studying various human diseases. In terms of hearing, the inner ear structure and hearing of the mini-pig have matured in the later stage of the embryo. Therefore, compared with other rodents and New Zealand rabbits, the mini-pig is unparalleled in establishing an animal model of human hearing loss. no advantage. Stem cell therapy for hearing loss, modeling of various inner ear diseases, and auditory electrophysiological experiments can be performed. The spiral ganglia, spiral edges, hair cells, and marginal cells of the cochlea's vascular striatum express Na + -K + -ATPase, and exhibit ion gradients of internal lymph and peripheral lymph as well as the stable function of each cell. It plays an important role in maintenance. Ouabain G (Ouabain) is an effective inhibitor of Na + -K + -ATPase, when combined with the α subunit of Na + -K + -ATPase, it inhibits the transport function of Na + and K + enzymes. possible. It increases the Na + concentration in the cell, inhibits Na + -Ca 2 + exchange, and increases the intracellular Ca 2 + concentration to generate positive muscle strength (to strengthen the heart). Ouabain can cause spiral ganglion neuron (SGN) damage in the inner ear of miniature pigs. The effect of the damage depends on the concentration and time. There is no obvious damage to the hair cells. Zhang Xueru et al. Toxicin G was used to establish a hearing loss model in miniature pigs.

　　(IVN) Auditory nerve (AN) after labyrinthectomy includes inferior vestibular nerve, superior vestibular nerve (SVN), cochlear nerve (CN), facial nerve (FN); B. Crypts and their signs: the 8th and 9th second cranial nerves and choroid plexus; C. ABI transplantation through maze simulation; D. CI simulation experiment; E. Cochlear stimulation electrode; F. The SCARA tympanic membrane window is used to simulate internal ear drug delivery via microfilaments (MF). Dura: Medulla, CP: Choroid plexus, BT: Brain tissue, SE: Stimulating electrode, C: Cochlea, RW: Round window, OW: Oval window, E: Medulla

　　1. Experimental methods Smiancin (0.2 ml/kg) and 2% pentopental sodium (1 ml/kg) were used to anesthetize Guizhou miniature pigs intramuscularly and placed in the left decubitus position, right ear, cheek and back of the neck. Prepare the skin, disinfect, and then spread out the surgical towel. Perform all surgical procedures in strict accordance with the requirements of aseptic surgery. Make a longitudinal incision along the posterior edge of the lower jaw, 1 cm from the edge of the ear and 1-2 cm at the angle of the upper jaw. The length of the incision is about 4-5 cm. It bluntly separates the subcutaneous and muscle tissue, exposing the stem and removing the attached tendons. , Remove the stem from the root to expose the milky white protrusions. Grind the mastoid inward with an electric drill, and then return it to the middle ear cavity, exposing the cochlear gyrus and round window niche. According to the experimental group, 200 L of vabein solution or normal saline gelatin sponge was placed in a circular window for 30 minutes. Then use a negative pressure aspirator to suck the fluid in the surgical cavity, suture the tissue layer after layer and close the surgical cavity and skin incision. After the operation, 1.6 million units of penicillin were injected intramuscularly every day for 7 consecutive days to prevent postoperative infection. Before the operation, 3 days after the operation, 1 week after the operation and 2 weeks after the operation, the hearing test of the animal ears was performed. Animals were anesthetized intramuscularly with smenoxin (0.2 ml/kg) and 2% sodium pentobarbital (1 ml/kg). After the animal is deeply anesthetized (steady breathing, heart rate of about 55-60 beats per minute, spontaneous muscle activity, corneal reflex appears), lie on your stomach and fix your limbs with your head in the center. We use

　　TDT to determine the ABR hearing threshold of the animal. The recording electrode is inserted at the midpoint of the connecting line between the front edge of the ear and the bone surface, the reference electrode is inserted subcutaneously into the ear canal of the test ear, and the ground electrode is inserted into the tip of the nose. Use "click to short an ear" stimulation, the duration is 100 milliseconds, the stimulation frequency is 21.1/s, and the collected signals are superimposed 1024 times. Earphones with built-in ear canals will emit sound. The low-pass filter is 50Hz and the high-pass filter is 3000Hz. The stimulation intensity starts from 90dB and gradually decreases in 5dB increments until the V wave disappears, and the ABR threshold is recorded. The maximum sound intensity of the audiometry equipment is 90 dB, so if the ABR waveform cannot be obtained at all 90 dB, the animal's hearing threshold is considered to be 100 dB. The Sig-Gen system records the hearing threshold of both ears.

　　2. The effect of Vaavein solution on spiral ganglia compared with the normal control group, the upper, middle and lower spiral ganglion cells in the 1 mM group were significantly reduced at 3, 1, and 2 weeks after surgery. .. Two weeks later, compared with the same group on the 3rd day after surgery, the damage of ganglion cells in the mural gyrus increased. However, compared with the normal control group, the number of upper and middle gyrus ganglia did not decrease significantly at 3 days and 1 week after surgery in the 0.1 mM group, but the number of lower gyrus ganglia did not decrease significantly. The number of lymph node cells decreases. Two weeks after the operation in the 0.1 mM group, compared with the normal control group, the number of spiral ganglion cells in the upper, middle and lower convolutions decreased, while the rotation of the upper, middle and lower convolutions decreased compared with 3 days after surgery. Spiral ganglion cells decrease. reduce.

　　3. The effect of Uavein solution on ABR is compared with the conventional control group. After the use of vabein, the ABRV wave threshold increases at 3 days, 1 week and 2 weeks after surgery. At 3 days, 1 week and 2 weeks after operation, the ABRV wave threshold was higher in the 1 mM group and 0.1 mM group. Compared with before the operation, the normal control group and the normal saline control group had no significant changes in hearing 2 weeks after the operation. There was no significant difference between preoperative and postoperative hearing in the normal control group. In each postoperative period, compared with the normal control group and the normal saline group, the ABRV wave threshold of the 1 mM group was significantly higher. On 3 days after operation, 1 week after operation and 2 weeks after operation, the ABRV wave threshold of 0.1 mM group was higher than that of normal control group and normal saline group. At 1 and 2 weeks after surgery, the ABRV wave threshold of the 1 mM group was significantly higher than that of the 0.1 mM group.