动物造模,帕金森病大鼠模型 z-bio 2021-12-30 1614

　　1. Modeling materials Animals: SD rats, females, 2 to 3 months old, weighing 180 to 220 g; drugs: 6-hydroxydopamine (6-hydroxydopamine, 6-OHDA), apomorphine, rabbit anti-tyrosine hydroxyl Avidin-biotin-peroxidase complex (Avidin-biotin-peroxidase complex, ABC) kit; equipment: rat brain stereotactic instrument, 307-4 benchtop dental drill cart, JEM100-SX Electron microscopy.

　　2. Modeling method The experimental animals were randomly divided into experimental group and control group, and the experiment was carried out after repeated behavior testing confirmed their non-rotating behavior:

　　(1) Experimental group: Rats were anesthetized by intraperitoneal injection of 1% pentobarbital (30mg/kg body weight), and the skull was placed in strict cranial position (the difference between the level of bregma and posterior bregma is less than 0.1mm, and the sagittal suture of the skull The horizontal height difference at 4mm is less than 0.1mm) is fixed on the rat brain stereotaxic instrument (note that the micro syringe needle handle is vertical to the operating plane), after routine disinfection, cut the skin and peel off the periosteum, refer to the rat brain stereotaxic Atlas, determine the two coordinates of the substanfia nigra compacta (SNC) and the nigrostriatal pathway on the left [①SNC posterior bregma (5.0±0.2) mm, and the left side of the sagittal suture (1.7±0.1) ) mm, (7.6±0.1) mm below the surface of the skull; ②(4.6±0.1) mm behind the Bregma of VTA, (0.9±0.1) mm on the left side of the sagittal suture, (7.5±0.1) mm below the surface of the skull]. Determine the specific coordinates of the rat’s weight and head size within the specified range and mark it with a triangular needle. Use a dental drill to carefully drill through the skull (be careful not to damage the dura). According to the determined coordinates, slowly insert the micro-injector to the predetermined depth, toward each of the two points. Inject 8μg of 6-OHDA (dissolved in 4μl of normal saline containing 0.2% ascorbic acid and prepare temporarily before use). The depth of injection should be the midpoint of the bevel of the needle hole as the reference point (take the dura mater under the skull as the 0 coordinate) Due to individual differences, the specific depth may vary slightly depending on the weight of the rat and the size of the head, and the injection can be moved within a certain range (the range should not exceed 0.3mm) to ensure that the 6-0HDA can be injected accurately. The injection speed is 10d/min, the needle is kept for 10min, and the needle is slowly withdrawn (2～3min). After the operation, the borehole is covered with dental glue, the wound is sutured routinely, and penicillin is injected into the intraperitoneal cavity for a week to prevent infection.

　　(2) Control group: Inject 4μl of normal saline (containing only 0.2% mass fraction of ascorbic acid) into the two coordinate points using the same method as above.

　　3. Modeling principle 6-OHDA is a norepinephrine homologue that selectively damages catecholamine neurons and their peripherals, and enables the synthesis of dopamine (DA) in the substantia nigra and its transport pathway to the striatum The damage caused the imbalance of catecholamine transmitters and acetylcholine transmitters, resulting in a series of characteristic changes similar to human Parkinson's disease (parkinson disease, PD) symptoms and pathology, such as lateral rotation.

　　4. General changes after modeling. Behaviour testing is started 1 week after the operation. Apomorphine (APO) is injected intraperitoneally (0.5mg/kg) to observe the changes in behavior. Once a week, the test will continue for 6 weeks. The rat constantly turns to the right and the rotation speed>7r/min is regarded as a successful PD rat model. If the rat does not rotate, the direction of rotation is not constant, the rat is constantly turned to the left, or the rat is constantly turned to the right but the speed is slower (< 7r/min), it is regarded as an unsuccessful PD model. In addition to rotating behavior, attention should also be paid to observe whether there are abnormal behaviors such as tremor, sluggishness, grasping, and sniffing.

　　5. Biochemical and pathological changes after modeling

　　(1) Immunohistochemical observation: The tyrosine hydroxylase (TH)-positive neurons in the substantia nigra of the left midbrain of PD model rats were significantly reduced compared to the right side, compared with the left and right side of the control group. The comparisons have extremely significant differences, but the right TH-reactive neurons in PD model rats have no significant differences with the control group. The TH-positive neurons in the successful PD model mice at 2 weeks, 3 weeks, 4 weeks, and 6 weeks after surgery were significantly reduced compared with 1 week after surgery, and 3, 4, and 6 weeks after surgery were also compared with 2 weeks after surgery. There are significant differences, but there is no significant difference between the TH-positive neurons at 3, 4, and 6 weeks after surgery.

　　(2) Electron microscopic observation: Under transmission electron microscopy, it can be seen that some neurons in the substantia nigra on the injured side of PD model rats are degenerated and necrotic, cell membranes are ruptured or incomplete, and nucleoli are ruptured. In some neurons, lysosomes increased in the cytoplasm, mitochondria and endoplasmic reticulum were slightly proliferated, and the nucleus pyknosis was not obvious, showing early apoptosis-like changes. Some neurons have dense chromatin or solidify into clear-bounded masses, some have organelle edema, unclear and shrunken cell membranes, electron dense deposits in the cytoplasm, and disappearance of lysosomes, showing late apoptotic appearance change.

　　6. Matters needing attention In the experiment, the weight of rats should be within the required range as much as possible, and the weight of 180-250g rats should be selected. Such rats have strong tolerance and their heads are easily fixed by the brain stereotaxic device; the substantia nigra target point To be sure, it should be carefully adjusted and verified according to the orientation map and stereotaxic instrument used; 6-hydroxydopamine should be freshly prepared, and the color should be orange-red; injection speed and needle retention are also very important; surgical instruments are strictly disinfected to prevent surgical infections. The trauma should be as small as possible, and the operation should be strictly aseptic; pay attention to the depth when drilling the skull, and stop immediately if there is a sense of breakthrough to prevent massive bleeding; the needle tip of the micro syringe should be slightly blunt to reduce the scope of drug penetration.

　　After the operation, the rats should be separated, and after awakening 3 to 5 hours, they can be kept in a cage to prevent the first awakening from licking and biting the wound of the unconscious rat. Keep the optimum temperature and humidity for the rats as much as possible in the breeding room.