A method for constructing a tree shrew epilepsy model

Abstract

The present invention discloses a method for constructing a tree shrew epilepsy model, which specifically includes the following steps: (1) preparation of Phrixotoxin‑1, dissolving 50 μg of Phrixotoxin‑1 in physiological saline, and adjusting the volume of physiological saline to 1 ml to prepare a solution with a concentration of 50 μg / ml, and a pH of 7.4; (2) injecting the Phrixotoxin‑1 solution prepared in step (1) into the tree shrew intraperitoneally, with an injection volume of 4 μg / kg. Tree shrews are small animals closely related to primates, with a high brain-to-body mass ratio, a relatively developed nervous system and a stress system similar to that of humans, and therefore have irreplaceable advantages over other non-primate animal models. The increased excitability of hippocampal pyramidal neurons in epileptic patients is due to the weakening of A-type potassium current (IA). In hippocampal and cortical neurons, IA is generated by Kv4 potassium channels. In the present invention, the specific peptide inhibitor of Kv4 potassium channel, Phrixotoxin-1, is used to prepare a tree shrew epilepsy model, providing a new method for preparing an epilepsy model.

Description

Technical Field

[0001] The invention belongs to the technical field of disease animal model construction, and in particular relates to a tree shrew epilepsy model construction method. Background Art

[0002] Epilepsy is a common brain disorder, commonly known as "epilepsy" or "epilepsy". It is characterized by persistent spontaneous epileptic seizures. Its incidence rate is second only to cerebrovascular disease. It is listed by the World Health Organization (WHO) as one of the five neuropsychiatric diseases that require global prevention and treatment.

[0003] The occurrence of epilepsy is a complex, multifactorial process. It is a symptom complex with multiple risk factors and strong genetic susceptibility. It is currently known that many factors such as heredity, abnormal brain development, trauma, infection, poisoning, and the interaction between environmental and genetic factors can lead to epilepsy. The latest research shows that the occurrence of epilepsy is not a single cellular or molecular event, but the result of abnormal development or destruction of the brain's homeostatic network system. The formation of abnormal neural networks facilitates the spontaneous discharge, diffusion and focus transfer of neurons, leading to the occurrence and recurrence of epilepsy. Therefore, it is particularly important to create an animal model with high similarity to human epileptic seizures. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing a tree shrew epilepsy model.

[0005] The object of the present invention is achieved as follows: (1) Phrixotoxin-1 is prepared by dissolving 50 μg of Phrixotoxin-1 in physiological saline, which is diluted to 1 ml to prepare a solution with a concentration of 50 μg / ml and a pH of 7.4;

[0006] (2) The Phrixotoxin-1 solution prepared in step (1) was intraperitoneally injected into tree shrews at an injection volume of 4 μg / kg.

[0007] The beneficial effects of the present invention are:

[0008] Tree shrews are small animals closely related to primates. They have a high brain-to-body ratio, a relatively developed nervous system, and a stress system similar to that of humans. Therefore, they have irreplaceable advantages over other non-primate animal models. The increased excitability of hippocampal pyramidal neurons in patients with epilepsy is due to the weakening of A-type potassium current (IA). In hippocampal and cortical neurons, IA is mainly produced by Kv4 potassium channels. In the present invention, Phrixotoxin-1, a specific peptide inhibitor of Kv4 potassium channels, is used to prepare a tree shrew epilepsy model, providing a new method for preparing an epilepsy model. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a comparison chart of HE staining results. DETAILED DESCRIPTION

[0010] The technical solution of the present invention will be described clearly and completely in combination with the embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, but not all of them. All other embodiments belong to the protection scope of the present invention.

[0011] , Experimental Animals

[0012] Twenty CL-grade male Burmese tree shrews weighing 120-150 g were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences [SCXK (Yunnan) K2023-0003] and kept in the ordinary animal room of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK (Yunnan) K2021-0003] in stainless steel cages at room temperature (24±2)℃, relative humidity (50±5)%, and light / dark cycle of 12h / 12h.

[0013] , Main Reagents

[0014] Reagent name company Phrixotoxin-1 alomone Paraformaldehyde recovery Hematoxylin and eosin (HE) staining kit Solebao

[0015] 3. Main instruments

[0016] Instrument Name company Rotary microtome German Leica Tissue spreading machine German Leica Paraffin embedding machine German Leica Freezer German Leica Automatic tissue dehydrator Wuhan Junjie Tissue slice baking machine Wuhan Junjie Holographic Scanner 3DHISTECH Electric heating blast drying oven thermocenter

[0017] 4. Preparation of drugs and reagents

[0018] Preparation of Phrixotoxin-1: 50 μg of Phrixotoxin-1 was dissolved in normal saline, and the volume of normal saline was adjusted to 1 ml to prepare a solution with a concentration of 50 μg / ml and a pH of 7.4.

[0019] , Experimental methods

[0020] 5.1 Animal model preparation

[0021] Experimental groups: ①, control group, ②, Phrixotoxin-1 (2μg / kg) group, ③, Phrixotoxin-1 (4μg / kg) group, ④, Phrixotoxin-1 (6μg / kg) group;

[0022] Group ① was intraperitoneally injected with the same amount of saline as group ③, and groups ②-④ were intraperitoneally injected with the corresponding phrixotoxin-1 to induce epileptic seizures. The behavioral performance of tree shrews was recorded within 2 hours.

[0023] 5.2 Obtaining tissue samples

[0024] 5.2.1 Acquisition of whole brain tissue

[0025] 24 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 tweezers, a triangular incision was cut from bottom to top, and the sternum and ribs were cut to expose the heart and liver. The perfusion needle was inserted into the left ventricle of the tree shrew, and the right auricle was cut at the same time, and PBS solution was perfused to allow blood to flow out quickly. After the liver and tongue of the tree shrew turned white, 4% paraformaldehyde was used for perfusion and fixation. When paraformaldehyde flowed to the brain, the tree shrew's tail would have a reflex phenomenon and the whole body would be stiff. At this time, the perfusion could be terminated, and the brain was removed after decapitation and placed in 4% paraformaldehyde for fixation.

[0026] 5.2.2 Acquisition of Hippocampal Tissue

[0027] 24 hours after modeling, the tree shrews were anesthetized, the skin on the tree shrew's neck was cut open, and then the skull was cut open. The skull was opened with curved forceps, and the brain was placed on an ice surface covered with a clean film, with the dorsal side facing up. The cortex was carefully opened and the hippocampus was taken out, and stored in a -80℃ refrigerator for later use.

[0028] 5.3 Behavioral assessment of epileptic seizures

[0029] The Racine score was used to evaluate the epileptic seizure behavior of tree shrews, with grade 0: no seizure behavior; grade 1: rhythmic twitching of the corners of the mouth, facial muscles or ears; grade 2: more severe local twitching; grade 3: clonus in the forelimbs, without upright or falling behavior; grade 4: generalized tonic-clonic behavior, accompanied by upright or lateral falling; grade 5: generalized tonic-clonic seizure, supine falling; grade 6: death. The tree shrews were observed continuously for 60 minutes, and the convulsion latency of the tree shrews was recorded, including the time from drug administration to the occurrence of grade 3 seizures (clonic latency) and the time from drug administration to the occurrence of tonic-clonic seizures (tonic latency), and the convulsion rate was recorded.

[0030] 5.4 HE staining

[0031] 5.4.1 Preparation of paraffin sections

[0032] (1) Dehydration: The tissue is still full of water after fixation. Since paraffin is a water-insoluble substance and cannot be mixed with water, a dehydrating agent must be used to remove the water in the tissue before wax immersion. This process is called dehydration. The steps are as follows: immerse in 75% ethanol for dehydration for 3 hours, immerse in 85% ethanol for dehydration for 1 hour, immerse in 90% ethanol for dehydration for 1 hour, immerse in 95% ethanol for dehydration for 1 hour, immerse in anhydrous ethanol for 1 hour, and replace anhydrous ethanol for another immersion for 1 hour.

[0033] (2) Transparency: Place in 50% anhydrous ethanol + 50% xylene for 50 min, then place in xylene for 40 min, and then replace with xylene for 50 min.

[0034] (3) Wax immersion: Place the transparent tissue block into melted paraffin at 58-60°C in an incubator, with three cups of wax in total. Wax the first cup for 1 hour, the second cup for 1.5 hours, and the third cup for 2 hours. This will allow the transparent agent xylene to be gradually expelled from the tissue, allowing the paraffin to penetrate more easily.

[0035] (4) Embedding: Pour the filtered new wax into the embedding container and place the wax-soaked tissue block in it as quickly as possible. Place the cut surface of the tissue block facing downward, leaving no gap between the tissue and the wax. Try to straighten and level the tissue and make it contact with the bottom plate of the embedding container as much as possible.

[0036] (5) Serial sectioning: Use a paraffin slicer to cut the paraffin block into 4 µm thick sections.

[0037] (6) Paste: The cut paraffin tissue slices are first placed in cold water for the first spread, then transferred to 45°C warm water for the second spread, and finally the tissue slices are picked up and attached to the slides coated with poly-lysine.

[0038] (7) Baking: Place the paraffin-sectioned slides on a slide rack and bake in an oven at 58°C for 1-2 hours.

[0039] 5.4.2 HE staining

[0040] (1) Paraffin sections were routinely dewaxed and hydrated: paraffin sections were dried at 60°C for 30 min, then immersed in xylene for 10 min, changed to xylene for 10 min, changed to anhydrous ethanol for 10 min, then immersed in 95% ethanol for 5 min, immersed in 90% ethanol for 5 min, immersed in 80% ethanol for 5 min, immersed in 70% ethanol for 5 min, immersed in distilled water for 5 min, and changed to distilled water for 5 min.

[0041] (2) Wash with distilled water for 5 minutes.

[0042] (3) Hematoxylin staining at room temperature for 3 minutes.

[0043] (4) Differentiation: Wash off the floating color with tap water, and differentiate with 1% hydrochloric acid alcohol (prepared by adding 1 ml of concentrated hydrochloric acid to 99 ml of 75% anhydrous ethanol) for 2 seconds.

[0044] (5) Reblueing: Rinse the slices in tap water for 15 minutes.

[0045] (6) Stain with eosin for 5 minutes at room temperature.

[0046] (7) Dehydration: Dehydrate by placing in graded alcohol (the concentration of graded alcohol increases by 5 percentage points from 75% to 100%) for 3 minutes each.

[0047] (8) Transparency: Place in xylene for 30 minutes.

[0048] (9) Seal the slides with neutral gum.

[0049] , Experimental results

[0050] 6.1 Experimental model preparation results

[0051] A total of 20 tree shrews (5 in each group) were used in this experiment. The tree shrews in the control group were normal, without convulsions or deaths; the convulsion rate of the Phrixotoxin-1 (2μg / kg) group was 20%, and there was no death; the convulsion rate of the Phrixotoxin-1 (4μg / kg) group was 100%, and the mortality rate was 20%; the convulsion rate of the Phrixotoxin-1 (6μg / kg) group was 100%, and the mortality rate was 80%. The results showed that the modeling effect of the Phrixotoxin-1 (4μg / kg) group was better.

[0052] Table 1 Tree shrew epilepsy model establishment

[0053] Grouping (n=5) Number of cases (only) Number of convulsions Seizure rate (%) Number of deaths mortality rate(%) Cause of death Control group 5 0 0.00% 0 0.00% / Phrixotoxin-1 (2 μg / kg) group 5 1 20.00% 0 0.00% / Phrixotoxin-1 (4 μg / kg) group 5 5 100.00% 1 20.00% Excessive convulsions Phrixotoxin-1 (6 μg / kg) group 5 5 100.00% 4 80.00% Excessive convulsions

[0054] 6.2 Evaluation of epileptic seizure behavior in each group of tree shrews

[0055] The seizure level of tree shrew epilepsy was evaluated according to the Racine score, and the results showed that the tree shrew epilepsy model induced by Phrixotoxin-1 was dose-dependent; with the increase of Phrixotoxin-1 dose, the clonic latency and convulsion latency of tree shrews became shorter, and the convulsion duration was longer.

[0056] Table 2 Behavioral assessment of epileptic seizures in tree shrews

[0057] Grouping (n=5) Clonic latency / s Seizure latency / s Seizure duration / s Phrixotoxin-1 (2 μg / kg) group 122.00±36.77 182 56 Phrixotoxin-1 (4 μg / kg) group 67.00±11.34 96.40±13.96 92.60±12.68 Phrixotoxin-1 (6 μg / kg) group 18.40±12.22 40.60±11.67 94.60±14.66

[0058] 6.3 Evaluation of epileptic seizure severity in each group of tree shrews

[0059] The seizure level of tree shrews was evaluated according to the Racine score. The results showed that there were 2 trees shrews with epileptic seizure level III or above in the Phrixotoxin-1 (2μg / kg) group; there were 5 trees shrews in the Phrixotoxin-1 (4μg / kg) group, but 1 of them died; there were 5 trees shrews in the Phrixotoxin-1 (6μg / kg) group, but 4 of them died.

[0060] Table 3 Seizure levels of tree shrews in each group

[0061]

[0062] 6.4 HE staining results

[0063] HE results showed that the structure of the neurons in the hippocampus of the tree shrews in the control group was intact and neatly arranged. The structure of the neurons in the hippocampus of the tree shrews in the Phrixotoxin-1 (2μg / kg) group was relatively intact and neatly arranged, with a small amount of cell necrosis. The hippocampus of the tree shrews in the Phrixotoxin-1 (4μg / kg) and Phrixotoxin-1 (6μg / kg) groups was severely necrotic, with chaotic arrangement of neurons and unclear edges.

Claims

1. A method for constructing a tree shrew epilepsy model, characterized in that: The steps include: (1) Preparation of Phrixotoxin-1: 50 μg of Phrixotoxin-1 was dissolved in normal saline, and the volume of normal saline was adjusted to 1 ml to prepare a solution with a concentration of 50 μg / ml, pH = 7.4; (2) The Phrixotoxin-1 solution prepared in step (1) was intraperitoneally injected into tree shrews at an injection volume of 4 μg / kg.

2. The method for constructing a tree shrew epilepsy model according to claim 1, characterized in that: The tree shrew is a CL-level Chinese-Burmese tree shrew.

3. The method for constructing a tree shrew epilepsy model according to claim 1, characterized in that: The tree shrew has a body mass of 120-150 g.

4. The method for constructing a tree shrew epilepsy model according to claim 1, characterized in that: The tree shrews are kept at room temperature of 24±2°C, relative humidity of 50±5%, and a light / dark cycle of 12h / 12h.

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