A method for modeling a tree shrew epilepsy model

Abstract

The application discloses a modeling method of a tree shrew epilepsy model, and specifically comprises the following steps: under a sterile condition, 1 mg of kainic acid powder is dissolved in 0.1 ml of sterile normal saline to prepare a 10 mg / ml kainic acid solution; under the sterile condition, 1 mg of N-methyl-D-aspartate powder is dissolved in 10 ml of sterile normal saline to prepare a 100 μg / ml N-methyl-D-aspartate solution; the tree shrew is subcutaneously injected in the neck with the prepared N-methyl-D-aspartate solution at a dose of 0.4 mg / kg, 12 hours later, the tree shrew is right brain ventricle injected at a dose of 60 μg / kg, and the modeling is completed after 24 hours. According to the application, the tree shrew is subcutaneously injected in the neck with 0.4 mg / kg N-methyl-D-aspartate 12 hours before kainic acid injection, so that a low-dose kainic acid (60 μg / kg) can produce an epilepsy-causing effect on the tree shrew, the epilepsy-causing rate is 100%, and no animal dies.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology for diseases, and particularly relates to a method for creating a tree shrew epilepsy model. Background Technology

[0002] Epilepsy is one of the most common chronic diseases of the central nervous system, second only to stroke and Alzheimer's disease in incidence. It is characterized by sudden, recurrent, and transient central nervous system dysfunction, a chronic brain disorder caused by excessive and abnormal discharge of brain neurons. It is a prevalent disabling neurological disease. Epileptic seizures are believed to be caused by an imbalance between excitatory and inhibitory neurotransmission. The pathogenesis of epilepsy is complex, with key mechanisms including abnormal neuronal discharge, apoptosis, neuroinflammatory responses, imbalance of excitatory and inhibitory neurotransmitters, and abnormal neural network remodeling. Excessive synchronous abnormal discharge of neuronal populations is a significant cause of epileptic seizures. Animal models play a crucial role in elucidating the basic mechanisms of epileptic seizures, identifying anti-epileptic drugs, and preventing the progression of epilepsy. Currently, epilepsy-related animal models mainly include chemically induced models, electrically induced models, and gene-editing models. Commonly used animals include rats, mice, and monkeys, with lower rodent models being the most common and widely used. Chemically induced models utilize rodents administered pentylenetetrazol (PTZ), kainic acid (KA), or pilocarpine systemically or intraventricularly. However, due to the distant relationship between rodents and humans, the animal models constructed from them differ significantly from human epilepsy in terms of pathogenesis and pathophysiological processes, making it difficult to directly apply experimental results to humans. Furthermore, primates are expensive and have high breeding costs, hindering their widespread use. Therefore, establishing epilepsy animal models that are more closely related to humans, have lower experimental costs, and can be widely used is particularly important. Summary of the Invention

[0003] The purpose of this invention is to provide a modeling method for a tree shrew epilepsy model.

[0004] The objective of this invention is achieved as follows: (1) Reagent preparation: Preparation of phycocyanin solution: Under aseptic conditions, dissolve 1 mg of phycocyanin powder in 0.1 ml of sterile physiological saline to prepare a 10 mg / ml phycocyanin solution; N-Methyl-D-aspartic acid: Under sterile conditions, 1 mg of N-methyl-D-aspartic acid powder was dissolved in 10 ml of sterile physiological saline to prepare a 100 μg / ml N-methyl-D-aspartic acid solution. (2) The N-methyl-D-aspartic acid solution prepared in step (1) was injected subcutaneously into the neck of the tree shrew at a dose of 0.4 mg / kg. After 12 hours, the solution was injected into the right ventricle of the tree shrew at a dose of 60 μg / kg. The model was completed after 24 hours.

[0005] The beneficial effects of this invention are: In this application, 0.4 mg / kg of N-methyl-D-aspartic acid was administered subcutaneously to the neck of tree shrews 12 hours before the administration of erythrocyanine. This resulted in a low dose of erythrocyanine (60 μg / kg) inducing epilepsy in tree shrews with a 100% epilepsy rate and no animal deaths. Attached Figure Description

[0006] Figure 1 Comparison of HE staining results in the hippocampus and cortex of tree shrews in five comparative experiments in this application; Detailed Implementation

[0007] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments are within the protection scope of the present invention.

[0008] Laboratory animals Twenty-five male Burmese tree shrews of ordinary grade, weighing 120-150 g, were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences [SCXK(Yunnan)K2023-0003] and housed in the ordinary grade animal facility of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK(Yunnan)K2021-0003]. They were housed in stainless steel cages at a room temperature of (24±2)℃, relative humidity of (50±5)%, and a light / dark cycle of 12 h / 12 ​​h.

[0009] Main reagents and consumables Reagent Name company Kainic acid (KA) Mce N-Methyl-D-aspartic acid (NMDA) Mce Hematoxylin and eosin (HE) staining kit Solarborg 3. Main Instruments Instrument Name company stereotaxic brain Rayward Micro-injection pump Lange Automatic dehydrator Wuhan Junjie Paraffin embedding machine Leica, Germany Rotary slicer Leica, Germany microscope OLYMPUS BX53 4. Preparation of reagents Kainic acid (KA): Under sterile conditions, dissolve 1 mg of KA powder in 0.1 ml of sterile physiological saline to prepare a KA solution of 10 mg / ml (10 μg / μl).

[0010] N-Methyl-D-aspartic acid (NMDA): Under sterile conditions, 1 mg of NMDA powder was dissolved in 10 ml of sterile physiological saline to prepare an NMDA solution of 100 μg / ml.

[0011] Experimental methods 5.1 Animal Model Preparation Experimental Groups: ① Normal (NC) group: 600 μl of sterile saline was injected subcutaneously into the neck, and 1 μl of sterile saline was injected into the right ventricle 12 hours later. ②, 60 μg / kg kaolin (KA) group: 600 μl of sterile saline was injected subcutaneously into the neck, and 12 hours later, a 10 μg / μl KA solution was injected into the right ventricle at a dose of 60 μg / kg. ③ Group with 80 μg / kg kaolin: 600 μl of sterile saline was injected subcutaneously into the neck, and 12 hours later, a ka solution with a concentration of 10 μg / μl was injected into the right ventricle at a dose of 80 μg / kg. ④ Group with 60 μg / kg kaolin (KA) + 0.2 mg / kg N-methyl-D-aspartic acid (NMDA): 0.2 mg / kg NMDA was injected subcutaneously into the neck, followed by a 10 μg / μl KA solution injected into the right ventricle 12 hours later at a dose of 60 μg / kg. ⑤ Group with 60 μg / kg kaolin (KA) + 0.4 mg / kg N-methyl-D-aspartic acid (NMDA): 0.4 mg / kg NMDA was injected subcutaneously into the neck, followed by a 10 μg / μl KA solution injected into the right ventricle 12 hours later at a dose of 60 μg / kg. 5.2 Injection method in the right ventricle (1) Fixing the tree shrew: The tree shrew was anesthetized by intraperitoneal injection of 3% sodium pentobarbital at 2 μl / g body weight. After complete anesthesia, the hair on the cranial face of the tree shrew was removed with animal clippers and fixed on the stereotaxic device. The ear rod was inserted into the tree shrew's ear canal and the left and right ear rods were balanced so that the line connecting the two ears of the tree shrew was in the same straight line as the ear rod. The tree shrew's incisors were pried open with tweezers and the upper incisors were locked in the hole of the adapter. The nose rod locking screw was pressed down and the height of the incisor clamp was adjusted up and down, and the position of the adapter was adjusted back and forth to keep the cranial face of the tree shrew horizontal. The screw was locked so that the tree shrew's head could not move. According to the tree shrew's body size, the tree shrew's body was raised with foam on the base of the stereotaxic device so that the head and body were kept horizontal to prevent the tree shrew's breathing from being blocked.

[0012] (2) Micro-injection pump parameter settings: Turn on the power switch of the micro-injection pump, set the operation mode to first draw and then infuse, and set the drawing parameters: the volume of liquid drawn, the drawing time is 0.5 min; the interval between drawing and infusing is 0.5 min; and the infusing time is 5 min.

[0013] (3) Positioning reference point: After fixing the RN type injection needle (specification 10μL) on the holder, turn on the power switch of the digital positioning instrument, disinfect the skin surface of the tree shrew skull with iodine, make a longitudinal incision from the line connecting the two ears to the eyes to expose the skull, find the intersection of the interauricular line and the sagittal suture, i.e. the anterior fontanelle point, use this point as the reference point of the three-dimensional coordinate system, move the three-dimensional manipulator arm to align its positioning point with the anterior fontanelle point, and press the "CLR" key of the digital display base X, Y, Z axis to zero (desktop digital positioning instrument).

[0014] (4) Locating the target area: Determine the coordinates of the right ventricle relative to the anterior fontanelle, namely ML value (X-axis), AP value (Y-axis), and DV value (Z-axis). Starting from the reference point of the anterior fontanelle, set the coordinate points according to the position of the right ventricle: AP: 2.0 mm in front of the anterior fontanelle, ML: 1.0 mm to the right of the midline. Move the operating arm the corresponding distance (absolute values ​​of AP and ML) to the target position (do not move the Z-axis). Then use the positioning pin as a reference point and mark it with a marker. Remove the cross-shaped operating arm and drill a hole in the skull.

[0015] (5) Experiment: After the micro-injection pump is turned on to draw the prepared composite solution, within 0.5 min, according to the determined value DV: 5.0 mm subdural, the operating arm is moved downward to the target depth. After the injection is completed, the needle is left in place for 5 min, and then the needle is slowly withdrawn at a speed of 1.0 mm / min. The coordinate value is set according to the method of locating the target area to carry out the injection experiment at another coordinate point. After the injection is completed, the RN-type injection needle is withdrawn, the injection hole is glued with bone glue, the wound is sutured and disinfected with povidone-iodine, and an appropriate amount of erythromycin ointment is applied to the wound. After the operation, penicillin (22000 IU / kg) is injected into the peritoneum.

[0016] Tissue sample acquisition 5.3.1 Acquisition of whole brain tissue Twenty-four hours after modeling, the tree shrew was anesthetized and fixed in a supine position on a foam board. The skin at the xiphoid process of the sternum was grasped with forceps, and a triangular incision was made from bottom to top. The sternum and ribs were then cut open to expose the heart and liver. A perfusion needle was inserted into the left ventricle of the tree shrew, and the right atrial appendage was simultaneously cut open. PBS solution was infused to allow rapid blood flow. After the liver and tongue turned white, the tree shrew was perfused and fixed with 4% paraformaldehyde. When the paraformaldehyde reached the brain, the tree shrew exhibited a tail reflex and generalized rigidity. At this point, perfusion was stopped, the head was severed, the brain was removed, and fixed in 4% paraformaldehyde for paraffin section preparation.

[0017] 5.3.2 Acquisition of hippocampal tissue Twenty-four hours after modeling, the tree shrew was anesthetized, the skin on its neck was cut open, the skull was cut open, the skull was opened with curved forceps, the brain was removed and placed on an ice surface covered with a clean film, dorsally facing up, the cortex was carefully lifted and the hippocampus was removed and stored in a -80℃ freezer for later use.

[0018] Epilepsy seizure behavior assessment The Racine scale was used to assess the seizure behavior of tree shrews. Grade 0: no seizure behavior; Grade 1: rhythmic twitching of the corner of the mouth, facial muscles, or ears; Grade 2: more severe focal twitching; Grade 3: clonic jerking of the forelimbs, without standing or falling; Grade 4: generalized tonic-clonic jerking, with falling while standing or lying on one's side; Grade 5: generalized tonic-clonic seizure, with falling while supine; Grade 6: death. Tree shrews were observed continuously for 60 minutes. The seizure latency was recorded, including the time from drug administration to the onset of a Grade 3 seizure (clonic latency) and the time from drug administration to the onset of a tonic-clonic seizure (tonic latency). The seizure rate was also recorded.

[0019] dyeing Paraffin sections were dewaxed to water and stained with hematoxylin at room temperature for 3-5 minutes. They were then washed with tap water for 10 minutes to remove excess stain, differentiated with 1% hydrochloric acid alcohol for 2 seconds, rinsed with tap water for 10 minutes, and observed under a microscope after the cell nuclei turned blue-purple. They were then stained with eosin at room temperature for 5 minutes, dehydrated in graded alcohols, cleared in xylene, mounted with neutral resin, and the color development results were observed and photographed under a regular optical microscope.

[0020] Experimental Results 6.1 Results of Experimental Model Preparation This experiment used 25 tree shrews (n=5). The control group showed normal results with no seizures or deaths; the 60 μg / kg KA group showed no seizures or deaths; the 80 μg / kg KA group had a 100% seizure rate and a 60% mortality rate; the 60 μg / kg KA + 0.2 mg / kg NMDA group had a 20% seizure rate and no deaths; and the 60 μg / kg KA + 0.4 mg / kg NMDA group had a 100% seizure rate and no deaths. The results show that NMDA can amplify the epileptogenic effect of KA, inducing epilepsy in tree shrews at lower doses of KA.

[0021] Table 1. Modeling details of the tree shrew epilepsy model Grouping (n=5) Number of examples (units) Number of seizures (individuals) Seizure rate (%) Number of deaths (animals) mortality rate(%) Cause of death NC Group 5 0 0.00% 0 0.00% / KA (60μg / kg) group 5 0 0.00% 0 0.00% / KA (80μg / kg) group 5 5 100.00% 3 60.00% Excessive and intense convulsions KA (60μg / kg) + NMDA (0.2mg / kg) group 5 1 20.00% 0 0.00% / KA (60μg / kg) + NMDA (0.4mg / kg) group 5 5 100.00% 0 0.00% / 6.2 Assessment of seizure behavior in tree shrews of each group The seizure severity of tree shrew epilepsy was assessed using the Racine score. The results showed that the combined use of NMDA and KA could induce tree shrew epilepsy even at low doses of KA, shorten the clonic latency, the seizure latency, and prolong the duration of seizures.

[0022] Table 2. Assessment of seizure behavior in tree shrews Grouping (n=5) Clonic latency / s Seizure latency period / s Seizure duration / s KA (60μg / kg) group none none none KA (80μg / kg) group 37.60±9.81 87.20±11.82 75.80±13.85 KA (60μg / kg) + NMDA (0.2mg / kg) group 123±19.80 194 32 KA (60μg / kg) + NMDA (0.4mg / kg) group 44.4±9.24 97.8±12.95 77.2±9.04 6.3 Assessment of the seizure severity in each group of tree shrews The seizure severity of tree shrews was assessed using the Racine score. The results showed that none of the 60 μg / kg KA group had a seizure severity of grade III or higher. All five birds in the 80 μg / kg KA group reached grade III, but three of them died. Two out of five birds in the 60 μg / kg KA + 0.2 mg / kg NMDA group reached grade III, and there were no deaths. All five birds in the 60 μg / kg KA + 0.4 mg / kg NMDA group reached grade III, and there were no deaths.

[0023] Table 3. Levels of seizures in tree shrews in each group. 6.4 HE staining results HE results showed that in the NC group, the hippocampal neurons of tree shrews had intact structure, were neatly arranged, had abundant cytoplasm, and clear nuclei. In the 60 μg / kg KA group, the hippocampal neurons of tree shrews were relatively neatly arranged and structurally intact, but a small number of neurons showed nuclear pyknosis and edema. In the 60 μg / kg KA + 0.2 mg / kg NMDA group, the hippocampal neurons of tree shrews were relatively neatly arranged and structurally intact, but the number of apoptotic neurons was higher than that in the 60 μg / kg KA group, and edema was present. In the 80 μg / kg KA group and the 60 μg / kg KA + 0.4 mg / kg NMDA group, the morphological and structural changes of some neurons in the hippocampus of tree shrews were obvious, with increased vacuoles, irregular cell arrangement, nuclear pyknosis, indistinct cytoplasm-nucleus boundaries, disappearance of some nuclei, and edema.

Claims

1. A method for establishing a tree shrew epilepsy model, characterized in that: Includes the following steps: (1) Reagent preparation: Preparation of phycocyanin solution: Under aseptic conditions, dissolve 1 mg of phycocyanin powder in 0.1 ml of sterile physiological saline to prepare a 10 mg / ml phycocyanin solution; N-Methyl-D-aspartic acid: Under sterile conditions, 1 mg of N-methyl-D-aspartic acid powder was dissolved in 10 ml of sterile physiological saline to prepare a 100 μg / ml N-methyl-D-aspartic acid solution. (2) The N-methyl-D-aspartic acid solution prepared in step (1) was administered subcutaneously to the neck of the tree shrew at a dose of 0.4 mg / kg. After 12 hours, erythrocyanine was injected into the right ventricle of the tree shrew at a dose of 60 μg / kg. The model was completed after 24 hours. The combined use of N-methyl-D-aspartic acid and erythrocyanine can induce epilepsy in tree shrews even at low doses of erythrocyanine.

2. The modeling method for the tree shrew epilepsy model according to claim 1, characterized in that, The specific method of right ventricular injection in step (2) is as follows: (1) Fixing the tree shrew: The tree shrew was anesthetized by intraperitoneal injection of 3% sodium pentobarbital at 2 μl / g body weight. After complete anesthesia, the hair on the cranial face of the tree shrew was removed with animal clippers and fixed on the stereotaxic device. The ear rod was inserted into the tree shrew's ear canal and the left and right ear rods were balanced so that the line connecting the two ears of the tree shrew was on the same straight line as the ear rod. The tree shrew's incisors were pried open with tweezers and the upper incisors were locked in the hole of the adapter. The nose rod locking screw was pressed down and the height of the incisor clamp was adjusted up and down, and the position of the adapter was adjusted back and forth to keep the cranial face of the tree shrew horizontal. The screw was locked so that the tree shrew's head could not move. According to the tree shrew's body size, the tree shrew's body was raised with foam on the base of the stereotaxic device so that the head and body were kept horizontal to prevent the tree shrew's breathing from being blocked. (2) Micro-injection pump parameter settings: Turn on the power switch of the micro-injection pump, set the operation mode to first draw and then infuse, and set the drawing parameters: the volume of liquid drawn, the drawing time is 0.5 min; the interval between drawing and infusing is 0.5 min; and the infusing time is set to 5 min. (3) Positioning reference point: After fixing the RN type injection needle on the holder, turn on the power switch of the digital positioning instrument, disinfect the skin surface of the tree shrew's skull with iodine, make a longitudinal incision from the line connecting the two ears to the eyes to expose the skull, find the intersection of the interauricular line and the sagittal suture, i.e. the anterior fontanelle point, use this point as the reference point of the three-dimensional coordinate system, move the three-dimensional manipulator arm to align its positioning point with the anterior fontanelle point, and press the "CLR" key of the digital display base X, Y, Z axes to zero; (4) Locating the target area: Determine the coordinates of the right ventricle relative to the anterior fontanelle, namely ML value, AP value, and DV value. Starting from the reference point of the anterior fontanelle, set the coordinate points according to the position of the right ventricle. AP: 2.0 mm in front of the anterior fontanelle, ML: 1.0 mm to the right of the midline. Move the operating arm the corresponding distance to the target position, and then use the positioning pin as a reference point and mark it with a marker pen; remove the cross-shaped operating arm and drill a hole in the skull. (5) Experiment: After the micro-injection pump is turned on to extract the prepared composite solution, within 0.5 min, according to the determined value DV: 5.0 mm below the dura mater, the operating arm is moved down to the target depth. After the injection is completed, the needle is left in place for 5 min, and then the needle is slowly withdrawn at a speed of 1.0 mm / min. The coordinate values ​​are set according to the method of locating the target area to carry out the injection experiment at another coordinate point. After the injection, the RN-type injection needle was withdrawn, the injection hole was sealed with bone glue, the wound was sutured and disinfected with povidone-iodine, and an appropriate amount of erythromycin ointment was applied to the wound. Postoperatively, 22,000 IU / kg of penicillin was administered intraperitoneally.

3. The method for establishing a tree shrew epilepsy model according to claim 1, characterized in that, The tree shrew in question is a common male Burmese tree shrew, weighing 120-150 g.

4. The modeling method for the tree shrew epilepsy model according to claim 1, characterized in that, The tree shrews were raised at a room temperature of 24±2℃, a relative humidity of 50±5%, and a light / dark cycle of 12h / 12h.

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