动物造模,药效评价,帕金森病 z-bio 2024-09-24 447

　　1. Modeling material animals: Healthy rhesus monkeys, male or female, weighing 3.7-5.5kg, aged 4-8 years old; Drug: N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), physiological saline, apomorphine.

　　2. The modeling method involves anesthetizing the animal and making a longitudinal incision of approximately 3cm upwards by 0.5cm above the sternotomy and along the midline of the neck. Separate the skin and subcutaneous tissue layer by layer, and bluntly separate and expose the bifurcation of the common carotid artery along the trachea at the inner edge of the sternocleidomastoid muscle. Separate the internal carotid artery and external carotid artery, and temporarily block the external carotid artery with a ligature. Extract fresh MPTP physiological saline solution at a dose of 1.2mg/kg, puncture the common carotid artery and inject slowly, completing the injection within 2 minutes. Release the blockage of the external carotid artery 2 minutes after needle removal. Press the puncture point for 5 minutes. After confirming no active bleeding, suture the skin. Inject 800000 U of penicillin intramuscularly and send it back to the feeding cage.

　　One week after surgery, a freshly prepared physiological saline solution of apomorphine was injected intramuscularly at a dose of 0.2mg/kg to induce rotational behavior. The successful evaluation criteria for the model in rhesus monkeys were rotation of 6 times/min or more towards the opposite side of the modeling surgery, increased muscle tone, decreased movement, and delayed response.

　　3. Modeling principle: MPTP itself does not have neurotoxicity, but after entering the brain, it is catalyzed by monoamine oxidase 8 in glial cells and serotonin neurons to be converted into 1-methyl-4-phenylpyridine (MPP), which then enters dopaminergic and noradrenergic neurons, inhibits mitochondrial ATP synthesis, and leads to chronic neuronal death.

　　4. Changes after modeling: Animals that were successfully modeled showed reduced eating and activity 5-7 days after surgery; The tension of the contralateral muscle increases during modeling, and contralateral limb tremors may occur (especially in the upper limbs); When sitting, support is needed, and when restraining the upper limbs, the body tilts towards the opposite side of the mold, or even tilts over; Symptoms such as dull expression and indifferent response to external stimuli. Symptoms such as reduced appetite, dull expression, and slow response to the outside world gradually improve around 2 weeks after the modeling surgery, while contralateral tremors often occur within 3 weeks after surgery and are rarely observed after 4 weeks. However, the increase in muscle tone and decrease in activity on the opposite side during modeling persist. One week after surgery, a freshly prepared physiological saline solution of apomorphine was injected intramuscularly. The animal showed symptoms of irritability and rotated 7-10 times/min towards the opposite side of the model. After 16 weeks of surgery, this method can still induce rotational movements of the same frequency.

　　5. Pathological and biochemical changes after modeling: Positron emission tomography (PET) was performed on the model animals one week before and 10 weeks after modeling, with the contralateral brain tissue used as a self control. Discovery of consistent metabolism on both sides before modeling; The metabolism of the basal ganglia on the modeling side decreased after surgery, which was significantly different from the control side. However, there was no significant difference in the metabolism of the control basal ganglia before and after modeling surgery.

　　Under the light microscope, it can be seen that the black matter structure on the modeling side is disordered, with a large number of cells missing, forming a honeycomb like structure. The control side structure is complete and clear. Immunohistochemical staining revealed that there were significantly more 7H (tyrosine hydroxylase) positive cells in the substantia nigra of the control side compared to the model side, with intact structures, regular cell bodies, clear cell processes, and obvious TH positive nerve fibers; However, the TH positive cells in the substantia nigra of the midbrain on the model side were significantly reduced, with irregular cell morphology, cell body shrinkage or disintegration, and inability to display cell protrusions, making it difficult to detect intercellular connections. The above changes are more pronounced in the outer central part of the dense zone of the substantia nigra than in the middle.

　　6. Precautions: This method requires surgical exposure of the internal carotid artery and temporary occlusion of the external carotid artery. Damaging the large arteries can cause fatal bleeding, requiring high surgical skills.