动物造模,药效评价,脑损伤 z-bio 2024-09-20 411

　　(1) Method of replication: Healthy rats, male or female, weighing 300-350g.

　　1) Falling impact device

　　① Organic glass tube: An organic glass tube with a length of 2m, an inner diameter of 19mm, and an outer diameter of 26mm, fixed at 3 points on the wall, with its lower end 15cm above the ground, for placing sponge beds and rats. Drill a small hole with a diameter of 5mm every 10cm on the pipe wall, corresponding to two rows, to reduce air resistance.

　　② Strike pad: a circular stainless steel plate with a diameter of 10mm and a thickness of 3mm, used to create a diffuse axonal injury model. Another method is to drill a small hole with a diameter of 1.5mm at a distance of 2.5mm from the center of the stainless steel pad, and place it in a stainless steel cylinder so that it is 3mm above the pad plane to create an animal model of diffuse brain injury combined with focal brain injury.

　　2) The method of brain injury in rats involves intraperitoneal injection of chloral hydrate (at a dose of 350-400 mg/kg body weight) or pentobarbital sodium (at a dose of 50-60 mg/kg body weight) for anesthesia. Skin disinfection in the surgical area, tracheal intubation, and assisted breathing with a ventilator. Femoral artery catheterization is used to record arterial pressure and monitor changes in electrocardiogram, blood pressure, pulse, respiration, etc. Fix the rat prone on a sponge bed with a known elastic coefficient, move it to the lower end of the organic glass tube, with the gasket facing the center of the organic glass tube, place a pulley above the organic glass tube, tie a small rope to the upper end of the copper column, and adjust the pulley until the rope crosses the pulley and the copper column is in the center of the organic glass tube. When the animal starts to wake up and has limb movements, drop a 400g heavy copper column from a height of 1m onto a stainless steel pad, immediately remove the sponge bed to prevent further damage, and move the rat to the operating table to receive respiratory support from a ventilator. Observe the respiration, heart rate, and blood pressure of animals during and before the impact. Record physical activity, urinary incontinence, changes in pupils, and neurological signs. After tapping, a time period of 3-24 hours can be selected to quickly decapitate the brain and record subarachnoid hemorrhage and visible brain contusions. And immerse the whole brain in formaldehyde solution for 24 hours, and make coronal sections of the fixed brain with a layer thickness of 2mm for HE staining. It can also be used for heart and brain perfusion. Tissue samples from the left and right parietal cortex, hippocampus, corpus callosum, brainstem, etc. are taken and processed into ultra-thin sections for electron microscopy observation.

　　(2) The mortality rate of animals after striking the model is around 25%.

　　1) The general symptoms after injury in all animals include vertical hair, urinary incontinence, limb twitching, unilateral or bilateral dilated pupils, respiratory depression, and other symptoms. Blood pressure immediately showed an increase in mean arterial pressure, followed by a period of hypotension, which returned to pre injury levels within 30-60 minutes. The heart rate rapidly slows down and gradually returns to pre injury levels after 10-15 minutes.

　　2) All traumatized mice showed obvious and extensive subarachnoid hemorrhage in the gross pathological changes. Scattered patchy hemorrhagic lesions were observed in the corpus callosum, upper arm of the cerebellum, brainstem, and other areas 3 hours after injury, presenting diffuse brain damage. Under low magnification, HE staining revealed significant subarachnoid hemorrhage. One hour after injury, a certain amount of axonal swelling and thickening were found on the immunohistochemistry stained sections of the brainstem, while HE staining did not show any changes in the axons during the same period; Immunohistochemical staining of the mouse brain at 3, 6, 12, and 24 hours after injury showed further changes in axonal injury in subcortical, corpus callosum, brainstem, and other areas, such as lobulated swelling, internal folding, and the formation of terminal and retractable spheres after axonal rupture. Swelling of neuronal cell bodies, dense staining of nuclei, superficial staining of cytoplasm, and reduction of Nissl bodies.

　　3) Electron microscopy observation shows separation, swelling, and internal folding of myelin sheaths, as well as disorder of nerve microfilaments within the axons and reduced microtubules in the transverse section. Myelin fibers often exhibit segmental degeneration and enlargement, disordered arrangement and degeneration fusion of nerve microfilaments, and loose onion like changes in myelin sheath layers. Partial dendrites also exhibit varying degrees of swelling, transparency, and vacuolization. Mitochondria swelling can be seen within glial cells, and the membranes before and after synapses are significantly thickened.

　　(3) In clinical comparative medicine, closed traumatic brain injury is mainly manifested as diffuse brain injury. This model simulates the characteristics of brain injury caused by external forces, and provides an external force to strike, causing significant changes in the animal's main vital signs. And the widespread intracranial hemorrhage, extensive damage to nerve tissue, and pathological changes in nerve cells are all similar to the diffuse damage characteristics of closed traumatic brain injury in humans. Moreover, with a fixed external force striking, the degree of damage to animals is similar and has good repeatability, which is conducive to the study of the pathological evolution process after injury and the judgment of treatment results.