CN110663646A - Chronic food-borne Parkinson disease mouse model and establishment method and application thereof - Google Patents
Chronic food-borne Parkinson disease mouse model and establishment method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of animal model establishment, and discloses a chronic food-borne Parkinson disease mouse model and an establishment method and application thereof.A 5% ferric citrate solution continuously intervenes in a C57BL/6 mouse for 16 weeks, behavioral evaluation is carried out by open field, accelerated rotation and pole climbing experiments every 4 weeks, and a suspension experiment and a food reward type Y maze experiment are carried out on the mouse at the 16 th week; the measured indexes of the total path, the average speed, the central times, the central path, the central time and the rod time of the mouse are obviously reduced compared with those of a control group, the turning time and the rod climbing time are obviously increased compared with those of the control group and are time-dependent, and the movement coordination, the balance force, the muscle tension and the activity of the mouse are obviously reduced. The invention simulates the clinic chronic morbidity condition of the Parkinson, and the chronic model is more suitable for monitoring the early change and screening the early diagnosis index of the Parkinson; has the characteristics of quantifiable behavioral disturbance and characteristic lesion loss of DA energy neuron degeneration.
Description
Technical Field
The invention belongs to the technical field of animal model establishment, and particularly relates to a mouse model for chronic food-borne Parkinson's disease and an establishment method and application thereof.
Background
Currently, the closest prior art: at present, existing PD models mainly comprise four types of chemical drug models, biological toxic substance models, protease inhibitor models and gene models, common MPTP mouse models, rotenone (rotenone) models, 6-hydroxydopamine (6-OHDA) models, dextran iron intraperitoneal injection models, FC (lateral ventricle injection) or ferric chloride models, gene knockout and other PD models basically belong to acute or subacute models, and the existing PD models have the defects that PD models are difficult to manufacture, behavioristics cannot be quantified, the acute reversibility, large side effects, part of animals are insensitive, the success rate is low and the like. Each molding method has different advantages and disadvantages, and the most suitable molding method needs to be selected according to the research purpose. Wherein the MPTP model is the most common model for researching PD at present due to economic and convenient model, MPTP is a neurotoxin which is nontoxic, but has high lipophilicity, can easily pass through the BBB of the body and then exist in cells such as 5-hydroxytryptamine neurons and glial cells, methyl-phenylpyridine ions (MPP +) are generated under the catalysis of monoamine oxidase B (MAO-B), the MPP + has high toxicity, it is released to extracellular space, taken up by DA transport protein, reversely transported to neuron cell body to cause selective damage to DA energy neuron, MPP + enters dopaminergic neuron, is actively taken up and gathered by mitochondria, specifically inhibits an electronic respiratory chain compound I in dopaminergic neuron mitochondria, reduces ATP generation to exhaust energy, generates a large amount of free radicals, and finally leads to the death of dopaminergic neurons. But the defects are that the degeneration loss of DA neurons is acute and reversible, the behavioral change of DA neurons can be recovered slowly along with time, the pathological damage and the behavioral change of DA of clinical PD patients are irreversible and more serious, no PD chronic pathogenesis process exists, and the characteristic change of PD diseases in early stage cannot be researched.
Parkinson's Disease (PD), also called parkinsonism, is a second major degenerative disease of the central nervous system, second to alzheimer's disease, mainly manifested by behavioral disorders such as resting tremor, bradykinesia, muscular stiffness, imbalance, and pathologically characterized by severe degeneration loss of dopaminergic neurons in the substantia nigra compacta and lewy bodies in remaining DA neurons in the brain. The incidence of PD is age-dependent, the incidence of middle-aged and elderly people is 0.3%, the incidence of the PD in the aged of 85 years old and older is 4.4%, the incidence of PD is younger at present, and the incidence of the PD is higher in men than in women. PD is high in morbidity and difficult to diagnose in early stage, dyskinesia and typical clinical symptoms can occur only when the dopaminergic neuron in substantia nigra is damaged by up to 70 percent, the physical health of the old is seriously damaged, the daily life of the old is influenced, and heavy economic burden is brought to families and society.
In the normal function of neurons, iron is an essential cofactor for the proteins involved, such as the required non-heme iron, tyrosine hydroxylase for the synthesis of catecholamine neurotransmitters, together with superoxide and H2O2The reaction generates highly reactive OH in chain reaction, and the OH and DA are oxidized to initiate the functions of neurotoxicity and the like. It was found that neuromelanin in DA-ergic neurons is the major neuronal storage site for iron, and that DA-ergic neurons are 4-fold more susceptible to free radical-mediated damage than total neurons. OH in PD-main source iron and H in Fenton reaction2O2The reaction produces the most toxic OH, which, in combination with endogenous antioxidant consumption, especially glutathione levels, can lead to increased oxidative stress and a dramatic decrease in glutathione content. So that the overload of iron promotes the decomposition of peroxide of lipid in brain to generate highly cytotoxic OH, destroys redox balance, and the electron transfer chain of mitochondria is disturbed, resulting in the gradual failure of endogenous respiratory function and the generation of energy metabolism disorder, thereby forming oxidative stress and threadsThe vicious cycle of mitochondrial damage eventually leads to excessive damage and loss of DA-neurons, resulting in death of DA-neurons, producing the clinical symptoms of PD.
Ferric Citrate (FC) has been used as a food nutritional additive, generally synthesized from ferric hydroxide and citric acid, chemically stable, and officially registered as an additive in the ministry of health and labour in japan on a designated list, and ferric citrate monohydrate has also been used as a commonly used additive in the united states and listed in federal regulations as a generally recognized substance in direct consumption, as an iron supplement commonly used in food, to supplement the body with iron elements in various foods such as flour or formula milk, and it has been demonstrated that FC absorption by the body increases brain iron content. The body's iron absorption and regulation is normally used in biological systems both in organic form (heme iron) and in inorganic form (ionic iron), the body extracting little iron from the diet (1-2 mg/day), but among them the diet trivalent iron (Fe)3+) Is the major source of total iron intake, so the absorbable iron supplement FC was chosen as the iron source for this model.
With the accelerated aging process of society, the increased incidence of PD will bring huge economic burden to society. The study of PD mechanism depends on the successful establishment of PD model, and the ideal PD model should show motor dysfunction and age-dependent DA-energy neurodegenerative pathology. In the past decades, toxin-based rodent models of PD have been essentially acute or subacute models, and although they have been widely used to study the pathogenesis and pharmacodynamics of PD, they have been necessary to establish a complete phenotypic animal model of PD because they rarely contain all the clinical behavioral disturbances and pathological features of PD, and have large side effects, difficult manipulation, and unsatisfactory results of research and evaluation.
In summary, the problems of the prior art are as follows: the existing PD model has the defects of difficult PD model manufacturing, reversible acute degree, large side effect, unquantized behavioristics, insensitivity of partial animals and low success rate.
The difficulty of solving the technical problems is as follows: since parkinson's disease is a chronic neurodegenerative disease of the nervous system, the MPTP mouse model is not consistent with the natural onset process of parkinson's disease. Since PD disease features include chronic and irreversible loss of DA-ergic neurons and changes in behaviours, although the current administration of large doses of MPTP to mice allows observation of pathological changes in dopamine damage, the potential for paralysis and even excessive death of the mice is not well studied at all to observe the process of degeneration of the substantia nigra and the progressive changes in behaviours. The small dose of MPTP is given acutely, the molding rate is low, the model time is short, the model is recovered gradually along with the prolonging of the time, the behavioral quantification is not completely met, and the MPTP mouse model can hardly solve the problems existing at present due to the limitation of the physiological mechanism of a mouse, so that a progressive dopaminergic neuron injury and progressive pathological changes are needed, and a chronic morbidity process is very important.
The significance of solving the technical problems is as follows: with the accelerated aging process of society, the increased incidence of PD will bring huge economic burden to society. The study of the PD mechanism relies on the successful establishment of a PD model, which should exhibit motor dysfunction, age-dependent DA-ergic neurodegeneration and abnormal α -Syn pathology. In the past decades, toxin-based rodent models of PD have been essentially acute or subacute models, and although they have been widely used to study the pathogenesis and pharmacodynamics of PD, they have been necessary to establish a complete phenotypic animal model of PD because they rarely contain all the clinical behavioral disturbances and pathological features of PD, and have large side effects, difficult manipulation, and unsatisfactory results of research and evaluation. The iron element participates in Fenton reaction to generate OS, and is closely related to PD, so that the study on whether the PD model can be successfully constructed by chronic food-borne iron overload is planned to be carried out, the problem existing in the construction of the current model is solved, the experiment adopts a long-term low-dose FC drenching mode to simulate the chronic iron overload condition, and the chronic iron overload condition is close to the PD clinical chronic morbidity condition to the maximum extent, so that a more scientific reference basis is provided for the construction of the PD model, and a reference is provided for the prevention, treatment, prognosis and mechanism study of the clinical PD diseases.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a mouse model of chronic food-borne Parkinson disease and an establishment method and application thereof.
The invention is realized in such a way that the establishment method of the chronic food-borne Parkinson disease mouse model comprises the following steps: the 5% ferric citrate solution intervenes in C57BL/6 mice continuously for 16 weeks, and behavioral evaluation is carried out every 4 weeks, including open field experiments, accelerated rotation experiments and pole climbing experiments, the mice at week 16 are subjected to suspension experiments and food reward type Y maze experiments, the total distance, the average speed, the central frequency, the central distance and the central time of the measured index mice are all obviously reduced compared with a control group, and the turning time and the pole climbing time are both obviously increased and are time-dependent compared with the control group.
Further, the establishment method of the chronic food-borne Parkinson disease mouse model adopts a 5% ferric citrate solution as a mouse Parkinson chronic model inducer.
Further, the mice of the establishment method of the chronic food-borne Parkinson disease mouse model are 9-month old and middle-aged mice.
The invention also aims to provide the chronic food-borne Parkinson disease mouse model established by the establishment method of the chronic food-borne Parkinson disease mouse model.
The invention also aims to provide application of the mouse model of chronic food-borne Parkinson disease in quantifiable behavioral disorders.
The invention also aims to provide application of the mouse model of chronic food-borne Parkinson disease in degeneration and loss characteristic lesions of dopaminergic neurons in substantia nigra pars compacta.
The invention also aims to provide application of the mouse model for chronic food-borne Parkinson disease in prevention, treatment, prognosis and mechanism of Parkinson disease.
The invention also aims to provide application of the mouse model for chronic food-borne Parkinson disease in monitoring early change and screening early diagnosis of Parkinson disease.
In summary, the advantages and positive effects of the invention are: according to the invention, ferric citrate is used for continuously intervening the 9-month-old C57BL/6 mice for 16 weeks, so that the mice are caused with behavioral disturbance and are time-dependent, the content of iron in substantia nigra is increased, and the degeneration loss of dopamine neurons is caused, a chronic food-borne mouse PD model is successfully established, the success rate of the established animal model is 30%, a foundation is laid for the PD animal model, and a new thought is provided for PD diagnosis and treatment. Because the incidence rate of PD in the elderly is high, the course of disease is long, the screening and detection of early PD markers are particularly important, the chronic PD model provided by the invention can be satisfied by long modeling time, and the model has advantages; besides, the model of the invention also has the characteristics of quantifiable behavioral disturbance and characteristic lesion caused by degeneration loss of DA energy neurons.
The invention adopts 5% ferric citrate (menstruum is physiological saline) to continuously intervene for 16 weeks in 9-month-old C57BL/6 mice, starts from the aspects of mouse behaviours, tissue iron content and pathological changes, explores whether chronic food-borne iron overload can construct a mouse PD model, and carries out behaviours evaluation method from the mouse gavage of 0-16 weeks, every 4 weeks to carry out behaviours experiments including an open field experiment, an accelerated rotation experiment and a pole climbing experiment, and carries out a suspension experiment and a food reward type Y maze experiment in the 16 th week. The content of the iron in the substantia nigra of the brain is increased, and the pathological changes mainly comprise the content of striatum dopamine DA, the content of substantia nigra lysine hydroxylase and the neuron changes of the striatum dopamine DA and the nigra lysine hydroxylase are obviously reduced, so that the success of a chronic food-borne mouse PD model is shown. The behavioural observation adopts the open field experimental device, the acceleration rotation experimental device and the pole-climbing experimental device which are provided by Jiangsu Sangson biotechnology limited company to complete the monitoring and the homemade suspension experiment and the food reward type Y maze experiment of Sichuan agriculture university to complete.
The invention provides a method for establishing a chronic food-borne mouse Parkinson disease model, which maximally approaches to the clinical chronic disease condition of PD, provides a more scientific reference basis for the construction of the PD model, and provides a reference for the prevention, treatment, prognosis and mechanism of clinical PD diseases.
According to the method for establishing the mouse model of the chronic food-borne Parkinson disease, the success rate of the established animal model is 30%, the reduction of DA (DA) neurons is observed in the prepared animal model, and the method is more suitable for monitoring early change and screening early diagnosis indexes of PD.
Drawings
FIG. 1 is a flow chart of a method for establishing a mouse model of chronic food-borne Parkinson's disease according to an embodiment of the present invention.
FIG. 2 is a graph showing the results of mouse body weight and daily food intake following FC intervention as provided by an embodiment of the present invention;
in the figure: (a) represents the weight results of FC-treated mice; (b) represents the results of daily food intake of FC-treated mice.
FIG. 3 is a schematic diagram showing the results of the FC intervention mouse open field experiment provided by the embodiment of the invention;
in the figure: (a) and (b) represents FC intervention mouse movement distance results; (c) and (d) represents FC intervention mouse mean velocity results; (e) and (f) represents FC intervention mouse median frequency results; (g) and (h) represents FC intervention mouse median time results; (i) and (j) represents FC intervention mouse median distance results; graphs (a), (c), (e), (g), (i) and (b), (d), (f), (h) and (j) represent graphs compared to the control group and not administered.
FIG. 4 shows the results of an FC intervention mouse spin-up experiment provided by an embodiment of the present invention;
in the figure: (a) and (b) represents FC intervention mouse at stick time results.
FIG. 5 is a schematic diagram of the results of a pole climbing experiment of a mouse with FC intervention provided by an embodiment of the present invention;
in the figure: (a) and (b) represents FC intervention mouse turn time results; (c) and (d) represents FC intervention mouse pole-climbing time results.
FIG. 6 is a schematic diagram of the results of FC intervention mouse suspension and food reward type Y maze experiments provided by the embodiment of the invention; represents comparison with control group.
FIG. 7 is a schematic diagram showing the change of the nigral iron content of a mouse subjected to FC intervention provided by the embodiment of the invention;
in the figure: (a) representing FC intervention mouse substantia nigra iron staining results; (b) intervention of mouse substantia nigra Fe for FC3+A mean optical density histogram; (c) represents the FC intervention mouse substantia nigra flame atomic method result.
FIG. 8 is a schematic diagram of the FC intervention mouse TH protein and gene level changes;
in the figure: (a) represents the mouse substantia nigra TH protein expression level after FC intervention; (b) represents the ratio of the number of TH positive cells of the mouse after FC intervention; (c) represents mouse substantia nigra TH gene expression level after FC intervention, and arrows indicate positivity.
FIG. 9 is a graph showing the results of the measurement of the content of DA and DOPAC in the striatum of a mouse intervened by FC according to the embodiment of the present invention.
FIG. 10 is a graph of the number of 5% FC-intervened mouse substantia nigra neurons compared to control provided by an embodiment of the invention;
in the figure: (a) FC intervenes in the nigrostriatal staining result of the mouse; (b) representing FC intervention mouse melanophore number change.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a mouse model of chronic food-borne Parkinson disease and an establishment method and application thereof, and the invention is described in detail below with reference to the accompanying drawings.
As shown in fig. 1, the method for establishing a mouse model of chronic food-borne parkinson's disease provided by the embodiment of the present invention comprises the following steps:
s101: continuously intervening C57BL/6 mice for 16 weeks by using 5% ferric citrate solution, wherein the mice are 9-month old and middle-aged mice;
s102: every 4 weeks carries out the behavioural evaluation, including open field experiment, rotation-accelerating experiment and pole climbing experiment, 16 th week mouse hangs experiment and food reward type Y maze experiment, and measured index mouse total distance, average speed, central number of times, central distance, central time compare the contrast group all obviously reduce in excellent time, turn time and pole climbing time compare the contrast group all obviously increase, and be the time dependence.
The technical solution of the present invention is further described below with reference to experiments.
Materials and methods
1.1 Experimental animals
60 SPF grade C57BL/6 mice at 36 weeks of age, supplied by Beijing Witongliwa laboratory animal technology Co., Ltd, production license: SCXK (jing) 2016-: SYXK 2014-. The animal welfare was in accordance with the "National Institutes of health guarde fortune and Use of laboratory animal" requirements, and the treatment of animals in the experiment was in accordance with the "guiding opinions on animals being treated" of the National institute of science and technology department of the people's republic of China. All experimental protocols were reviewed and approved by the animal welfare and use committee of the university of Sichuan agriculture.
1.2. Establishment of chronic food-borne PD mouse model
1.2.1 Ferric Citrate (FC) solution formulation and administration
The FC (from Doxolone) solution is prepared at present, and the FC and normal saline ratio is as follows: FC [ m (FC): V (physiological saline) ═ 2.5g:50mL ], was heated to boiling to dissolve, and the mice were gavaged uniformly at 10 am daily for 16 weeks.
1.2.1 weight and feed intake monitoring, 5% FC intervention mice for 16 weeks, mice weight and feed intake were weighed weekly.
1.2.2 behavioral assessment
1.2.2.1 open field experiments
A mouse open field experimental device (model SA215) consists of four reaction boxes, an automatic data acquisition device and an ANYmaze behavior video analysis system, and is provided by Jiangsu Saeons Biotechnology limited. The length, width and height of the reaction box in the open field experiment of the mouse are both 50cm, the inner wall and the bottom surface are white (the color is opposite to the hair of the mouse), and the bottom surface of the reaction box is averagely divided into 25 squares (5cm multiplied by 5cm) by using software. The automatic data collector is used 2m above the centers of the four reaction boxes, and the collection visual field of the automatic data collector can cover the four reaction boxes (the collection process keeps a certain position). The mouse is placed in the center of the bottom surface in the reaction box, and the automatic data collector records the movement of the mouse for 10 min. And cleaning the inner wall and the bottom surface of the reaction box by using alcohol so as to prevent the urine, the excrement and the smell remained by the previous animal mouse from influencing the test result of the next mouse. The noise of the experimental environment is controlled below 65dB, the illumination is kept consistent, the interference to the movement of the mouse is reduced as much as possible, and the stress reaction of the animal is prevented. Parameters including total distance of movement of the mice, average speed, number of centers, time of center, and distance to center are available from the american ANYmaze animal behavior video analysis system. Every 4 weeks, one mouse.
1.2.2.2 accelerated spin test
The mouse accelerated rotation experimental device (model SA 102) consists of a rotating lane and a control system and is provided by Jiangsu Saeons Biotechnology Co. The mice were placed on a constant speed rotating rod (rotating speed 5r/min), lane width 9cm, rotating rod diameter 3 cm. After the mice stabilized, they underwent an incremental increase of 1RPM every 8 seconds with an acceleration of 7r/min2, a maximum speed of 40r/min, a test time of 5min, and the time on the stick was recorded for the mice. The test was performed every 4 weeks, three consecutive tests were performed per mouse and the average value was taken, and each mouse received 3 training sessions in advance the day before the official test.
1.2.2.3 Pole climbing experiment
The pole climbing experimental device (model is SA225 type) is provided by Jiangsu Saeons biotechnology limited, and the model is SA225 type mouse pole climbing experimental device. The device is composed of a wood rod and a base, wherein the wood rod is 1cm in diameter and 50cm in height, and an anti-skid wood ball with the diameter of 3cm is arranged at the top of the wood rod. The wooden pole is vertically placed, the head of the mouse is upward, the wooden pole is vertically placed on the ground, the mouse naturally climbs down along the pole, the turning time of the body of the mouse rotating 180 degrees and the pole climbing time of the mouse climbing to the bottom of the wooden pole are recorded, and the average value is obtained through five times of continuous tests. Once every 4 weeks, one day before each test, training mice climbed from the top to the bottom of the pole, and each animal received 3 training sessions in advance.
1.2.2.4 suspension experiments
The mouse double forelimbs are hung on an iron wire with the diameter of 1.5mm and the distance from the ground of 30cm, the mouse is prevented from overturning by covering the iron wire 1cm above the rod, the time before landing is recorded, the interval of each test is 1min, and the average value is obtained after 5 tests.
1.2.2.5 food reward type Y maze experiment
The mouse food rewarding Y-maze experiment contained two test phases (1 h apart). Mice were fasted the day before testing, and the mice were kept starved the next day. The 1 st stage is the training period, the new arm is blocked by the baffle, the mouse is put into the maze by the initial arm and freely moves for 10 min. After 1h, the 2 nd stage detection period is started, the mouse is placed into the labyrinth from the original initial arm, the new different arm partition plate is pulled out, the mouse feed is placed on the new different arm, and the mouse freely moves in the labyrinth for 5 min. The camera recorded the number of times the mouse shuttles on each arm within 5min of mouse activity.
In the test, the mice were uniformly arranged to match the three arms of the initial arm, the new arm and the other arms of the Y maze, and the three arms were fixed at two stages of the maze experiment. And cleaning the inner wall and the bottom surface of the reaction box by using alcohol so as to prevent the urine, the excrement and the smell remained by the previous animal mouse from influencing the test result of the next mouse. The noise of the experimental environment is controlled below 65dB, the illumination is kept consistent, and the interference to the movement of the mouse is reduced as much as possible.
1.2.3 detection of iron content in brain tissue
1.2.3.1 flame atomic method
Weighing about 5mg of midbrain (substantia nigra) tissues in a digestion tube, adding 10 ml of concentrated nitric acid, putting the mixture into a microwave digestion tank for digestion for 2 hours, performing acid dispelling operation until the liquid in the digestion tube is soybean in size, cooling to room temperature to fix the volume, and measuring on a flame atomic absorption instrument.
1.2.3.2 iron staining
After brain tissue was fixed with 4% paraformaldehyde, washed overnight with running water, then dehydrated with gradient alcohol, and embedded in wax block after toluene was transparent. Wax alignment using a paraffin slicerThe blocks were sliced consecutively with a slice thickness of 5 μm. And (2) immersing the paraffin section into dimethylbenzene and gradient alcohol for dewaxing, rehydrating with distilled water for 3min, and immersing the section in 7% potassium ferrocyanide and 3% hydrochloric acid in a dark place according to the weight ratio of 1: 1 Perls solution for 30min, washed 3 times with PBS, 5min each, and washed with 1% H2O2Soaking in distilled water for 30min, washing with distilled water for 3 times, each time for 3min, adding 0.25mg/mL DAB and 0.02% H2O2The obtained product is developed in PBS for 10min, counterstained with sappan wood for 10s, dehydrated by gradient alcohol, transparent in xylene, sealed with neutral gum, and observed under an optical microscope.
1.2.4 brain histology examination
1.2.4.1 Nie staining
Paraffin sections are sequentially subjected to xylene transparency and gradient alcohol dewaxing, then are soaked in distilled water for 2min, a Niger dye solution (prepared by toluidine blue) is dyed at 37 ℃ for about 8min, the paraffin sections are washed for 1 time by distilled water, alcohol gradient dehydration is carried out, the sections are sealed after the xylene transparency, neurons are observed under an optical microscope, and counting is carried out by using Image pro plus software.
1.2.4.1 immunohistochemistry
Baking paraffin sections in a 60 ℃ oven for 1H, dewaxing by xylene and gradient alcohol, rehydrating with distilled water, and adding 3% H2O2The mixture was left in the dark for 20min, the endogenous catalase was removed, and the mixture was washed 3 times with PBS for 5min each. And (3) placing the slices into a sodium citrate buffer solution, performing microwave repair for 5min, repeating for 3 times, and washing for 3 times by PBS after the liquid is naturally cooled. Blocking 5% BSA at room temperature for 1h, diluting primary antibody TH (ENZO, USA, 1: 1000), dripping onto tissue, and keeping away from light at 4 deg.C overnight to allow antibody antigen to bind sufficiently. After washing with PBS for 3 times, the biotinylated secondary antibody was added dropwise and incubated at room temperature for 1 h. After washing with PBS for 3 times, adding streptavidin-peroxidase complex dropwise, and incubating for 1h at normal temperature. Washing with PBS for 3 times, rinsing with distilled water for 1 time, dripping DAB prepared freshly onto the tissue, developing for 5-10 min, observing under a microscope, and placing in distilled water to terminate the reaction. Gradient alcohol dehydration, xylene transparency, neutral gum sealing, and observation under an optical microscope.
1.2.4 striatal dopamine content detection
Mouse Dopamine (DA) ELISA test kit, mouse 3, 4-dihydroxyphenylacetic acid (DOPAC) ELISA test kit (purchased from shanghai enzyme-linked biotechnology limited) were used and the procedures were strictly followed.
Second, result in
2.1 As in FIG. 2, 5% FC intervention was 16 weeks, and there was no significant change in mouse body weight and feed intake compared to the control group, indicating that the mice had essentially no stress during molding.
2.2 as in FIG. 3, 5% FC intervention 16 weeks, open field test results in mice as follows
2.2.15% FC intervention 16 weeks, total mouse movement distance decreased
As shown in fig. 3a and 3b, the total course of 5% FC intervention mice was significantly reduced compared to the control group from 4 weeks to 16 weeks of administration (P < 0.01 or P < 0.05), and significantly reduced from 4 weeks onwards over the course of administration compared to no administration (P < 0.01).
2.2.25% FC intervention 16 weeks, mice mean velocity of movement decreased
As shown in fig. 3c and 3d, the mean rate of 5% FC intervention mice was significantly reduced from 12 weeks and 16 weeks of administration compared to the control (P < 0.01 or P < 0.05), and significantly reduced from 8 weeks to 16 weeks over the time of administration compared to no administration (P < 0.01).
2.2.35% FC intervention 16 weeks with reduced number of centers of mouse movement
As shown in fig. 3e and 3f, the median frequency of 5% FC interventions in mice was significantly reduced compared to the control group (P < 0.01 or P < 0.05) from 8 weeks to 16 weeks of administration, and significantly reduced compared to no administration (P < 0.01) from 8 weeks to 16 weeks over the course of administration time.
2.2.45% FC intervention 16 weeks with reduced central range of mouse movement
As in fig. 3g and 3h, the 5% FC intervention mouse central course was significantly decreased from week 8 until week 16 of dosing (P < 0.01) compared to the control group, and significantly decreased from week 8 to week 16 over the dosing time (P < 0.01) compared to no dosing.
2.2.55% FC intervention 16 weeks with a decline in mouse motor median time
As shown in fig. 3i and 3j, the 5% FC intervention mice were significantly decreased in median time from week 8 to week 16 of dosing (P < 0.01) compared to the control group, and significantly decreased from week 8 to week 16 with the passage of dosing time (P < 0.01).
2.35% FC intervention 16 weeks, mice decreased in bar time
As shown in the figures (FIGS. 4a and 4b), 5% FC intervention mice were significantly reduced in bar time (P < 0.01 or P < 0.05) from week 4 to week 16 after administration compared to the control, and were significantly reduced in bar time (P < 0.01) from week 4, week 12 and week 16 after administration compared to no administration, and were reduced in turn and climb time from week 2.45% FC intervention for week 16
As in figures 5a and 5b, 5% FC intervention mice had significantly increased turn times compared to the control group from week 4 to week 16 of dosing (P < 0.01), and significantly increased over the dosing time at weeks 4, 8 and 16 (P < 0.01 or P < 0.05). The 5% FC intervention mice had significantly increased pole-climbing time compared to the control group from week 4 to week 16 of dosing (P < 0.01), and significantly increased with the passage of dosing time at weeks 4, 8, and 16 (P < 0.01).
2.55% FC intervention 16 weeks later, mice hang time and frequency of entry into neobrachium decreased
As shown in FIGS. 6a and 6b, the suspension time of the 5% FC-intervened mice was significantly reduced compared to the control group (P < 0.01), and the frequency of the mice entering the new arm was reduced compared to the control group (P < 0.05).
After 2.65% FC intervention for 16 weeks, the content of nigral iron in mice is obviously increased
As shown in FIG. 7, the results of the iron staining method (panels a and b) and the flame atomic method (panel c) showed that the iron content of the substantia nigra of the 5% FC-intervened mice was significantly increased (P < 0.05 or P < 0.01) compared to the control group.
2.65% FC intervention 16 weeks later, mice increased TH protein and gene level expression
As shown in fig. 8a and 8b, 5% FC intervention mouse TH exhibited a significant reduction in substantia nigra pars compacta neuronal cytoplasm (P < 0.01) compared to the control group, as shown in fig. 8c, with gene and protein levels consistent.
After 2.65% FC intervention for 16 weeks, the striatal DA and DOPAC content of the mice is obviously increased
As shown in FIG. 9, 5% FC intervention mice have significantly reduced striatal DA and DOPAC levels (P < 0.01) compared to the control.
2.65% FC intervention 16 weeks later, the number of black neurons in mice decreased
As shown in fig. 10a and 10b, the number of black neurons in 5% FC-intervened mice was significantly reduced compared to the control group (P < 0.01).
The results of the behavioral analysis, the iron content change and the histopathology prove that the chronic food-borne mouse PD model is successfully modeled. Under the condition of 16 weeks of 5% FC intervention, the SN iron content of the mouse is increased, dopaminergic neurons are lost, the motor activity and the motor capacity of the motor behavior of the mouse are obviously reduced, the time dependence is realized, and the successful establishment of the chronic food-borne PD mouse model can simulate the clinical practical slow pathogenesis process and mechanism of PD, has longer modeling time compared with the acute and subacute PD models, and is more suitable for monitoring the early change of PD and screening the early diagnosis index of PD.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (8)
1. The method for establishing the chronic food-borne Parkinson disease mouse model is characterized by comprising the following steps of: the 5% ferric citrate solution intervenes in C57BL/6 mice continuously for 16 weeks, and behavioral evaluation is carried out every 4 weeks, including open field experiments, accelerated rotation experiments and pole climbing experiments, the mice at week 16 are subjected to suspension experiments and food reward type Y maze experiments, the total distance, the average speed, the central frequency, the central distance and the central time of the measured index mice are all obviously reduced compared with a control group, and the turning time and the pole climbing time are both obviously increased and are time-dependent compared with the control group.
2. The method for establishing the chronic food-borne Parkinson disease mouse model according to claim 1, wherein a 5% ferric citrate solution is used as a mouse Parkinson chronic model inducer.
3. The method for constructing a mouse model of chronic food-borne Parkinson's disease according to claim 1, wherein a 9-month-old aged mouse is used as the mouse in the method for constructing a mouse model of chronic food-borne Parkinson's disease.
4. A chronic food-borne Parkinson's disease mouse model established by the establishment method of the chronic food-borne Parkinson's disease mouse model according to any one of claims 1-3.
5. Use of the mouse model of chronic food-borne Parkinson's disease according to claim 4 for quantifying behavioral disorders.
6. Use of the mouse model of chronic food-borne Parkinson's disease according to claim 4 for the pathological changes characterized by degenerative loss of dopaminergic neurons of the substantia nigra pars compacta.
7. Use of the mouse model of chronic food-borne Parkinson's disease according to claim 4 for the prevention, treatment, prognosis, and mechanism of Parkinson's disease.
8. Use of the mouse model of chronic food-borne Parkinson's disease according to claim 4 for monitoring early stage changes and screening for early diagnosis of Parkinson's disease.
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