动物造模,药效评价,慢性视神经损伤 z-bio 2024-08-07 395

　　1. Modeling material animals: Adult healthy domestic cats, male or female, weighing 2.5-3.5kg; Drug: Anesthetic; Equipment: Experimental balloon.

　　2. Modeling method: Surgical approach mimicking human wing points. After intraperitoneal injection of 3% pentobarbital at a dose of 1ml/kg to anesthetize animals, they were placed in a lateral position with their limbs fixed and their head tilted to one side. An intravenous infusion channel was established, and 400000 U penicillin was added intravenously to 100ml of physiological saline. Prepare skin in the surgical area, disinfect with povidone iodine, take the line between the outer canthus and the tragus as the surgical incision, about 3cm long, cut open the scalp, aponeurosis, bluntly separate the muscles, peel off the periosteum, drill and remove the skull, and enlarge the bone window to a size of 2cm x 2cm. Under a surgical microscope, the dura mater is incised in a star shape to the bone margin, and cerebrospinal fluid is removed. The temporal lobe brain tissue is slowly lifted and penetrated along the sphenoid spine until white optic nerve and optic chiasm are seen. Implant a non removable balloon in the lateral gap of the optic nerve, followed by a catheter approximately 20cm in length, which is led through a subcutaneous tunnel to the neck and back. Inject 0.1ml of contrast agent and check that the balloon is located between the optic chiasm of the optic nerve and the underlying skull base bone structure. Heat and seal the end of the catheter, and after checking for leaks, bury the catheter subcutaneously in the neck and back. Pay attention to observing vital signs and neurological deficits after surgery. Normal group: No surgical treatment was performed; Fake surgery group: perform craniotomy surgery and insert a balloon without filling.

　　3. The modeling principle adopts the balloon implantation method to cause chronic optic nerve injury and establish a chronic compression injury model of the optic nerve.

　　4. After modeling, the animals can survive normally without fever, infection, epilepsy, or limb movement disorders. The CT scan shows that the balloon position is normal and there is no leakage.

　　The latency of F-VEPI waves and wave II in the normal group and sham surgery group, as well as the peak to peak values of waves I-II, remained relatively constant, while the rest of the waveforms showed significant variation among individuals. Four weeks after chronic compression of the cat's optic nerve, F-VEP begins to show significant abnormalities, characterized by a low, wide, and flat waveform, prolonged latency periods I and II, decreased peak to peak values of I-II, and the abnormalities become more pronounced with prolonged compression time.

　　5. After modeling, the average density of retinal ganglion cells labeled with Dil retrograde in the normal group was (465 ± 38) cells/mm, with a higher density in the central area than in the peripheral area. There was no significant change in the number of RGCs in the first 4 weeks after optic nerve compression, but there was a significant decrease in the number of RGCs by 8 weeks after compression, with an average density of (293 ± 32)/mm, a decrease of about 37%.

　　Under light microscopy, retinal HE staining: Normal adult cat retina HE staining shows three layers of nuclei, with clear boundaries between each layer. From the vitreous cavity to the sclera, they are the ganglion cell layer, bipolar cell layer, and photoreceptor cell layer. Node cells can be clearly divided into two types of cells based on nuclear morphology: one type is large nucleated cells, with lighter staining and sometimes visible nucleoli inside the nucleus; The other type has smaller nuclei and deeper staining. Four weeks after chronic compression of the optic nerve, the changes in the retina were still not significant. Eight weeks after compression, the RGC nuclei were significantly sparse, and the large and lightly stained nuclei disappeared. The total thickness of the retina became thinner, and glial cells proliferated.

　　Optic nerve bright red 2R bright green staining: Normal optic nerve myelin sheath appears pink, while axons, nerve bundle membranes, and inner membranes appear green; Degenerated myelin sheaths do not stain due to the disappearance of phospholipids. The normal arrangement of optic nerve myelin sheaths is neat and dense. After 4 weeks of compression, pink twisted strands and local staining loss are observed, and the local staining loss worsens after 8 weeks of compression.

　　Electron microscopy examination: ① Retina: Normal cat RGC nuclei are oval in shape, with large nuclei and uniform nucleoplasm. Sometimes nucleoli or nuclear membrane invagination can be seen, and the proportion of cytoplasm to cells is small, but the organelles are abundant. There was no significant change in the first 2 weeks after compression, but at 4 and 8 weeks after compression, the cytoplasmic components of RGCs were found to be loose, the endoplasmic reticulum expanded, mitochondria swelled, vacuoles appeared under the plasma membrane, nuclear membrane wrinkled and sunken, chromatin edge aggregation, and cytoplasmic cavitation Optic nerve: The myelin sheath structure of normal cat optic nerve is intact, with clear and tightly arranged myelin plates, intact axon membrane, and uniformly distributed microfilaments and microtubules in the axon. Two weeks after compression, only slight demyelination was observed, and the arrangement of the cytoskeleton was mildly disrupted. Four weeks after compression, there was delamination of the lamina, vesicular dissociation of myelin sheaths, vacuolar appearance of axons, disordered arrangement of microfilaments and microtubules, swelling of mitochondria in the cytoplasm, widening of cristae spaces, and blurred cristae. At 8 weeks, significant thinning of the myelin sheath and regeneration of myelin sheath can be observed.

　　6. Precautions: Before the experiment, check that the refractive interstitium of both eyes of the animal is clear, the pupils are equally large and round, the light response is good, and there are no abnormalities in the fundus. In order to ensure the smooth success of the surgery and the normal survival of the postoperative animals, it is important to follow the principle of aseptic operation, promptly replenish fluids, and administer antibiotics during the surgery to prevent infection. Avoid accidental damage to the optic nerve, optic chiasm, and their supply vessels. Inject contrast agent in batches to gradually enlarge the balloon, in order to simulate the clinical pathological process of chronic optic nerve injury as much as possible. When filling the contrast agent, it should be injected slowly, and regular CT scans must be performed after surgery to ensure that the balloon position is normal and there is no contrast agent leakage.