A method for constructing a bumblebee model of acute or chronic neuropathy

Abstract

The present invention discloses a method for constructing a bumblebee model of acute or chronic neuropathy. The method uses newly emerged bumblebees as the subjects for establishing the bumblebee model of neuropathy; acrylamide is dissolved in a sterile sucrose solution to prepare an acrylamide sucrose solution; the newly emerged bumblebees are fed the acrylamide sucrose solution to obtain the bumblebee model of neuropathy; the model constructed by the method of the present invention can be used to screen methods or drugs for preventing or treating neurotoxicity-related diseases, can be applied to explore the specific mechanisms of nervous system-related diseases, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal model construction, and particularly relates to a method and application of constructing a bumblebee model of acute or chronic neuropathy by inducing acrylamide. Background Art

[0002] Acrylamide (ACR) is a chemical reagent widely used in various industrial processes (such as wastewater purification and ore processing) and molecular experimental research. It is a recognized neurotoxin and a possible human carcinogen. Repeated exposure to acrylamide in humans or animals can cause peripheral neuropathy, characterized by muscle weakness, ataxia, even limb paralysis, and axonal degeneration. Acrylamide's harmful effects on humans primarily manifest as potential neurotoxicity, genotoxicity, reproductive toxicity, and carcinogenicity. Long-term, low-dose exposure to acrylamide can cause symptoms such as drowsiness, mood and memory changes, hallucinations, and tremors, accompanied by peripheral neuropathy. Acrylamide can also cause cognitive impairment, damage Kenyon cells (KC cells), and lead to genetic changes and chromosomal abnormalities, inducing tumors and cancer.

[0003] Early studies have shown that acrylamide-induced neurotoxicity may be associated with damage to nerve terminals in the peripheral and central nervous systems. Morphological, electrophysiological, and electrochemical studies have demonstrated that nerve terminals are the primary targets of acrylamide toxicity. Furthermore, acrylamide can attack thiol-containing protein sites, interfere with the presynaptic nitric oxide (NO) signaling pathway, damage presynaptic nerve terminals, disrupt neural signaling, and produce neurotoxicity. Acrylamide can induce apoptosis in various neural cells, including human neuroblastoma SH-SY5Y cells, astrocytoma U1240-MG cells, and rat stellate cells, potentially impairing brain homeostasis and causing neurotoxicity. Acrylamide-induced neurotoxicity is typically manifested by intracellular glutathione depletion, which mediates mitochondrial dysfunction and oxidative stress, resulting in oxidative stress damage to the brain. Acrylamide can cause circadian rhythm impairment and spatial memory impairment in mice by downregulating the expression of circadian rhythm proteins. It also increases intestinal permeability, reduces tight junction protein expression, and increases lipopolysaccharide levels in the intestine and serum, inducing immune inflammatory responses, disrupting tight blood-brain barrier junctions, damaging neurons, and leading to memory impairment. Currently, the specific mechanisms of acrylamide neurotoxicity and its prevention and treatment remain unclear, especially the neurotoxic effects of acrylamide at different exposure doses and durations, which require further research and testing. Therefore, establishing animal models is an important adjunct to studying the mechanisms and prevention strategies of acrylamide neurotoxicity.

[0004] Animal neurotoxicity testing has long been a fundamental component of nonclinical laboratory assessments. Early studies often used mammals such as primates, rodents, or canines as test animals for acrylamide neurotoxicity. While these mammals have their own advantages, they also have disadvantages such as lengthy model development cycles and high costs. Furthermore, the neurotoxicity and carcinogenicity of acrylamide can cause pathological changes and, in many cases, death, resulting in high mortality rates, making these models highly unstable and limiting their application. As an alternative, the development of non-rodent animal models such as zebrafish, Drosophila melanogaster, and Caenorhabditis elegans has become an innovative platform for testing and screening the neurotoxic effects of acrylamide. Among these non-traditional model organisms, bumblebees, as social insects, share similarities in their nervous system and behavior with humans, making them effective models for studying acrylamide neurotoxicity. In addition, bumblebees have the advantages of good ecological representativeness, behavioral sensitivity, biological characteristics and low ethical risks, and have good learning and cognitive abilities, including olfactory learning, color discrimination and recognition and operational behavior of specific targets. They are a good animal model for studying the neurotoxicity caused by acute and chronic toxic concentrations of acrylamide. Summary of the Invention

[0005] The present invention provides a method for constructing a bumblebee model of acute or chronic neuropathy by inducing acrylamide, which can be used to screen drugs for preventing or treating acute or chronic neuropathy, and can also be used to explore the mechanisms of diseases related to acute or chronic neuropathy.

[0006] The method for constructing the bumblebee model of acute or chronic neuropathy of the present invention is as follows:

[0007] Animal selection: Bumblebees on the first day of burrowing were selected as subjects for establishing the bumblebee model of neuropathic injury.

[0008] Preparation of acrylamide sucrose solution: First, prepare a sterile sucrose solution with a mass volume concentration (g:mL) of 45-55%. After sterilization, add acrylamide to the sterile sucrose solution to prepare an acrylamide sucrose solution with a concentration of 1.5-2.5mmol / L or 0.1-0.15mmol / L.

[0009] Model establishment: Newly emerged bumblebees were fed with 1.5-2.5 mmol / L acrylamide solution for 2 days to obtain bumblebees with acute neuropathy; newly emerged bumblebees were fed with 0.1-0.2 mmol / L acrylamide sucrose solution for 5 days to obtain bumblebees with chronic neuropathy.

[0010] The present invention tests whether a bumblebee model of neuropathy induced by acute toxicity or chronic toxicity is successfully constructed by measuring behavioral indicators; the behavioral indicators include measurement of bumblebee movement ability and measurement of bumblebee learning and memory ability.

[0011] After reaching the corresponding feeding days, the present invention analyzed the movement distance, movement speed, maximum acceleration and minimum acceleration of the bumblebees in the corresponding acrylamide acute toxicity and chronic toxicity groups through video software.

[0012] After reaching the corresponding feeding days, the present invention tests the learning level and memory ability of bumblebees in the corresponding acrylamide acute toxicity and chronic toxicity groups to odors through a beak extension experiment.

[0013] The present invention has the beneficial effect of establishing an acrylamide neurotoxicity model using bumblebees fed different acrylamide doses and durations. Both acute and chronic acrylamide exposure reduce the bumblebees' motor ability and impair their cognitive abilities, demonstrating acrylamide neurotoxicity. Compared to other common animal models in the prior art, the present invention is simple to operate, low-cost, and has a short modeling cycle. It exhibits significant neurotoxicity, with varying acute and chronic neurotoxicity depending on the dose and duration of exposure. It is suitable for large-scale neurotoxicity testing and can be used to screen for methods or drugs to prevent or treat neurotoxicity-related diseases. It can also be applied to explore the specific mechanisms of nervous system-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Results of the movement distance (A), movement speed (B), maximum acceleration (C) and minimum acceleration (D) of bumblebees in the chronic group during the video time;

[0015] Figure 2 This is the crawling trajectory curve of bumblebees in the chronic group;

[0016] Figure 3 Results of the movement distance (A), movement speed (B), maximum acceleration (C) and minimum acceleration (D) of bumblebees in the acute group during the video time;

[0017] Figure 4 This is the crawling trajectory curve of bumblebees in the acute group;

[0018] Figure 5 This is the learning curve of the chronic group in the bumblebee proboscis extension experiment;

[0019] Figure 6 This is the memory of the odor in the chronic group 3 hours after the bumblebee proboscis extension test;

[0020] Figure 7 This is the learning curve in the acute group bumblebee proboscis extension experiment;

[0021] Figure 8 This is the memory of the odor in the acute group of bumblebees 3 hours after the proboscis extension experiment. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment.It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the scope of the present invention.It should be understood by those skilled in the art that, without departing from the spirit and scope of the present invention, the details and forms of the technical solutions of the present invention can be modified and replaced, but these modifications and replacements all fall within the protection scope of the present invention.Similarly, for clarity and conciseness, the description of well-known functions and structures is omitted in the following description. Example 1

[0023] 1. Acrylamide (analytical grade) was purchased from BioDee Biotechnology Co., Ltd. 500 g of sucrose was weighed and diluted to 1 L with deionized water. The solution was sterilized by filtration using a 0.22 μm filter membrane in a vacuum filtration flask to obtain a sterile sucrose solution with a mass volume concentration of 50%. Acrylamide was dissolved in the sterile sucrose solution to prepare 2 mmol / L and 0.1 mmol / L acrylamide sucrose solutions.

[0024] The pollen was divided into ziplock bags, tightly sealed, and sterilized by electron beam sterilization at an intensity of 20-40 kGy for 3-5 hours to obtain sterile pollen.

[0025] 2. Place unemerged honeycomb spleens in a plastic box (17 × 11.5 × 6.7 cm) filled with 50% sucrose solution. Punch a hole in the top of the box and insert a 2 mL centrifuge tube filled with sterile sucrose solution. Place the box in a constant temperature incubator at 65% humidity and 29°C to observe the maturation of the pupae. One day after incubation, observe the birth of new bees in the honeycomb spleens. Select healthy bumblebees that have emerged from the nest and have a chest width of 5 ± 0.5 mm and carefully remove them. Place three bumblebees in a transparent culture cup for subsequent experiments.

[0026] 3. Set up a motor ability test group and a learning and memory test group. For the motor ability test group, randomly select 48 bumblebees selected in step 2 and divide them into an acute group and a chronic group, with 12 bumblebees in each experimental group and 12 bumblebees in the control group.

[0027] For the learning and memory test group, 120 bumblebees selected in step 2 were randomly selected and divided into an acute model group (60 bumblebees) and a chronic model group (60 bumblebees), with 30 bumblebees in each experimental group and 30 bumblebees in the control group.

[0028] Acute experimental group: bumblebees were given free access to 2 mmol / L acrylamide-sucrose solution and sterile pollen for 2 days;

[0029] Chronic experimental group: bumblebees were given free access to 0.1 mmol / L acrylamide-sucrose solution and sterile pollen for 5 days;

[0030] Control group: Bumblebees were allowed to freely eat sterile sucrose solution and sterile pollen.

[0031] Example 2: Determination of bumblebee motility

[0032] Individual bumblebees from the acute and chronic groups were placed in petri dishes in a quiet, well-lit room at room temperature. After the bees acclimatized, they were videotaped for 30 minutes without disturbing their normal movements or maintaining their current environment. The videos were then imported into Etho Vision software to delineate each bumblebee's movement area and label the bees within that area. The distance, speed, maximum acceleration, and minimum acceleration of each bumblebee during the video were analyzed. Changes in movement patterns were also analyzed in bumblebees exposed to short-term high-dose and long-term low-dose acrylamide exposure compared to controls fed a normal sterile sucrose solution diet.

[0033] The results of the chronic group experiment are shown in Figure 1 、 2 , Figure 1 The results showed that feeding 0.1mmol / L acrylamide sucrose solution for 5 days significantly reduced the four motor indicators of bumblebees and weakened their motor ability. Figure 2 The crawling trajectory curve shows that the control group actively crawled on the entire board, while the acrylamide experimental group tended to move at the junction and showed a certain circular curve.

[0034] The results of the acute group experiment are shown in Figure 3 、 4 , Figure 3 The results showed that compared with the control group, the movement distance, average speed, maximum acceleration and minimum acceleration of bumblebees were significantly reduced after feeding with 2mmol / L acrylamide sucrose solution for 2 days. Figure 4 The crawling trajectory curve shows that in the control group, Xiong Feng was more inclined to crawl across the entire board, with denser crawling trajectories, while the crawling trajectories after feeding acrylamide were sparser, and Xiong Feng was more inclined to move in a circular motion along the junction of the boards.

[0035] Example 3: Determination of bumblebee learning and memory abilities

[0036] 1. For bumblebees that have reached acute toxicity or chronic toxicity levels, remove the bumblebee diet in advance before testing and starve the bumblebees for 2 hours;

[0037] 2. Place the bumblebee into a fixture (a processed 2mL centrifuge tube). Clamp the bumblebee's neck with an insect pin. Secure the ends of the insect pin with silicone ear plugs. Then, use cut insulating tape to secure the ends of the insect pin to the 2mL centrifuge tube. Label the tube wall. Then, place a water-soaked cotton ball at the bottom of the 2mL centrifuge tube to prevent the bumblebee from drying out. Place the observation device on a tray and label it in order.

[0038] 3. Behavioral observation room: A completely dark room with a constant light source and a room temperature of 26°C. It is equipped with an exhaust fan to prevent odor accumulation.

[0039] 4. Before learning and memorizing, place the fixed bumblebees in a behavioral observation room and keep them still. Turn on the air conditioner to maintain the room temperature so that the bumblebees can get familiar with the environment and avoid stress.

[0040] 5. Use a clean needle to poke holes in the clean filter paper (the more the better), then fold the filter paper in half and insert it into a 20mL syringe. Use a 2mL syringe to draw 0.5mL of lemon essential oil and inject the liquid onto the filter paper from the front end of the 20mL syringe until the liquid is completely absorbed by the filter paper.

[0041] 6. Before starting the learning process, test the bumblebees with 50% sugar water to see if they have a proboscis extension reaction. If they do, then you can start the scent learning process. If most of them do not, then continue to keep them in a hungry state.

[0042] During the learning process, place a single bumblebee in front of an exhaust fan, aim a clean 10 mL empty syringe at the front of the bumblebee's antennae, and give air at a constant speed for 5 seconds. Then, aim a lemon syringe at the front of the bumblebee's antennae and give odor at a constant speed for 10 seconds. The shorter the interval between the two air administrations, the better. Give sugar 6 seconds after giving the lemon odor (do not touch the antennae, but let the bumblebee eat some sugar water every time. Be careful not to give too much to prevent it from not extending its beak after eating). The above is the process of learning once. There needs to be an interval of 10 minutes between each bumblebee's learning times, and 10 rounds of learning are repeated. Each time, record whether the bumblebee extends its beak after giving sugar. If the beak extension reaction occurs more than 3 seconds after giving sugar, do not record it.

[0043] 7. Long-term memory test (tested 3 hours after learning the odor): Use lemon odor to expose the bumblebees for 10 seconds, and record whether they react to the lemon odor. Then use 50% sugar water to test whether the bumblebees have a proboscis reaction and record it. This is used to determine whether the bumblebees do not extend their proboscis because they are not hungry.

[0044] The results of the chronic group experiment are shown in Figure 5Compared with the control group, the experimental group showed no proboscis reaction to the lemon essential oil stimulation in the first five rounds of testing. However, they showed some memory of the lemon essential oil odor in the sixth round of stimulation, and the memory eventually stabilized at 37.93%. After a 3-hour memory period, the bumblebees' response to the lemon essential oil odor was measured, and it was found that 37.93% of the bumblebees in the experimental group extended their proboscises upon stimulation, indicating memory of the odor. This was significantly different from the 68.75% memory rate in the control group ( Figure 6 );

[0045] The results of the acute group experiment are shown in Figure 7 In the second round of lemon essential oil stimulation, the bumblebees showed a beak extension effect, and then the learning rate continued to increase, finally stabilizing at 36.67%. The memory test results 3 hours later showed that the final memory rate of the bumblebees was still 36.67%, which was significantly lower than the 62.16% of the control group, indicating that short-term high-dose acrylamide caused damage to the bumblebee's learning level and memory ability ( Figure 8 ).

[0046] The above results show that the method of the present invention can successfully construct a bumblebee model of acute or chronic neuropathy, and the present invention provides a new model for the study of neurotoxicity-related diseases.

Claims

1. A method for constructing a bumblebee model of acute or chronic neuropathy, characterized in that: It includes the following steps: Animal selection: Newly emerged bumblebees were selected as subjects for establishing the bumblebee model of neuropathic injury; Prepare acrylamide sucrose solution: dissolve acrylamide in sterile sucrose solution to prepare acrylamide sucrose solution; Model establishment: Newly emerged bumblebees were fed acrylamide-sucrose solution to obtain a bumblebee model with neuropathic effects; When the mass volume concentration of sterile sucrose solution is 45-55% and the concentration of acrylamide in the sucrose solution is 1.5-2.5 mmol / L, newly emerged bumblebees are fed for 2 days to obtain bumblebees with acute neuropathy; When the mass volume concentration of the sterile sucrose solution is 45-55% and the concentration of acrylamide in the sucrose solution is 0.1-0.15 mmol / L, newly emerged bumblebees are fed for 5 days to obtain bumblebees with chronic neuropathy.

2. Use of the bumblebee model of acute or chronic neuropathy constructed by the method for constructing the bumblebee model of acute or chronic neuropathy according to claim 1 in screening drugs for preventing and / or treating acute or chronic neuropathy.

3. Use of the bumblebee model of acute or chronic neuropathy constructed by the method for constructing the bumblebee model of acute or chronic neuropathy according to claim 1 in studying the disease mechanism of acute or chronic neuropathy.

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