Methods for constructing animal models

Abstract

The application relates to the field of animal model construction, in particular to an animal model construction method. The application provides application of an NF-kappa B inhibitor in animal model construction. The NF-kappa B inhibitor PDTC provided by the application is mainly concentrated in the inflammation field in previous research, and has not been applied in epilepsy model construction. The method for constructing an animal model by using PDTC provided by the application can construct an ideal epilepsy model which is low in price, simple in operation, stable in onset and clear in the epileptogenic process, and is helpful to further expand new mechanisms of epilepsy onset and evaluate effects of antiepileptic drugs, and is a new application of the pharmacological tool drug PDTC.

Description

Technical Field

[0001] The present invention relates to the field of animal model construction, and in particular to a method for constructing an animal model. Background Art

[0002] Epilepsy is a chronic neurological disorder characterized by recurrent seizures and caused by abnormally synchronized neuronal discharges in the brain. Globally, approximately 68 million people suffer from epilepsy, with 20-40% suffering from intractable epilepsy. Currently, the pathogenesis of epilepsy remains largely unexplained. Therefore, establishing stable and reliable animal models of epilepsy is essential for exploring the disease's pathogenesis and screening anti-epileptic drugs.

[0003] Currently, the most widely recognized animal models of epilepsy include the febrile seizure model, the kainic acid-induced epilepsy model, the lithium chloride-pilocarpine-induced epilepsy model, the pentylenetetrazol-induced epilepsy model, and the 4-aminopyridine-induced epilepsy model. With the in-depth study of various epilepsy models, researchers have discovered that each model has its own unique epileptogenic mechanism. Consequently, they have proposed multiple hypotheses about the pathogenesis of epilepsy, which has greatly promoted the development of anti-epileptic drugs. However, due to the complexity of the human epilepsy process, a large proportion of patients still cannot be explained by existing theories. Therefore, the development of new epilepsy models to further explore new epileptogenic mechanisms is crucial for the prevention and treatment of epilepsy. At the same time, many existing epilepsy models also have many disadvantages such as high cost, complex operation, and large individual response differences. Therefore, the search for a low-cost and easy-to-administer pharmacological tool drug to construct a stable animal model of epilepsy has important scientific significance and application value. Summary of the Invention

[0004] In light of this, the present invention provides a method for constructing an animal model. Using the classic NF-κB inhibitor Ammoniumpyrrolidinedithiocarbamate (PDTC) as a tool drug, the present invention successfully constructed a novel epilepsy model mouse via intraperitoneal injection. This method provides a simple, inexpensive, and stable model that can be widely applied to epilepsy pathogenesis and anti-epileptic drug screening research, with promising application prospects.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of an NF-κB inhibitor in constructing an animal model.

[0007] In some embodiments of the present invention, the NF-κB inhibitor in the above application includes PDTC.

[0008] In some embodiments of the present application, the animal model in the above-mentioned application is an epilepsy animal model.

[0009] The present application also provides a method for constructing an animal model, which comprises administering an NF-κB inhibitor to a rodent.

[0010] In some embodiments of the present application, the method for constructing an animal model further comprises a detecting step.

[0011] In some embodiments of the present application, the method for constructing an animal model, the detecting comprises: behavioral detection and pathological detection; the behavioral detection comprises one or more of seizure grade, latency, duration or survival rate; the pathological detection comprises hematoxylin-eosin staining or immunohistochemical staining.

[0012] In some embodiments of the present application, the rodent in the method for constructing an animal model is raised in a standard environment, with free access to water and food.

[0013] In some embodiments of the present application, the rodent in the method for constructing an animal model comprises a mouse.

[0014] In some embodiments of the present application, the mouse in the method for constructing an animal model is a 7-8 week old C57BL / 6 mouse.

[0015] In some embodiments of the present application, the method for constructing an animal model comprises intraperitoneal injection.

[0016] In some embodiments of the present application, the method for constructing an animal model comprises administering a dose of 150 mg / kg / d.

[0017] The present application also provides an animal model obtained by the above-mentioned method for constructing an animal model.

[0018] The present application also provides the use of the animal model obtained by the above-mentioned method for constructing an animal model in exploring the pathogenesis of epilepsy and / or evaluating anti-epileptic drugs.

[0019] The present application also provides the use of the animal model obtained by the above-mentioned method for constructing an animal model in preparing a drug for preventing or treating epilepsy.

[0020] The present application provides the use of an NF-κB inhibitor in constructing an animal model.

[0021] The NF-κB inhibitor PDTC provided by the present application is mainly concentrated in the field of inflammation in previous studies, and has not been applied to the construction of epilepsy models. The method for constructing an animal model using PDTC described in the present application can construct an ideal epilepsy model with low price, simple operation, stable onset and clear epileptogenic process, which is helpful for further expanding new mechanisms of epilepsy and evaluating the effects of anti-epileptic drugs, and is a new application of the pharmacological tool drug PDTC. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0023] Figure 1 Figure 2 shows the behavior of epileptic mice after intraperitoneal injection of PDTC; A shows the seizure grade; B shows the onset latency; C shows the duration; D shows the survival rate;

[0024] Figure 2 Figure 3 shows the brain tissue pathology of epileptic mice after intraperitoneal injection of PDTC; A shows hematoxylin-eosin staining; B shows immunohistochemical staining of microglial marker IBA1 and astrocyte marker GFAP. DETAILED DESCRIPTION

[0025] The invention discloses a method for constructing an animal model.

[0026] It should be understood that the expression "one or more of" includes individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

[0027] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0028] It should be understood that the order of steps or the order in which certain actions are performed are not important as long as the present invention remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0029] The use of any and all examples or exemplary language, such as "such as" or "including," herein is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0030] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant numerical values ​​in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, all ranges, amounts, values, and percentages used in this disclosure should be understood to be modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specified value or range.

[0031] In one aspect, the present invention provides a novel method for preparing a drug-induced epilepsy animal model, comprising the following steps:

[0032] Step 1: Select normal rodents, raise them in a standard environment, and give them free access to food and water. Divide the animals into two groups: a control group and a model group.

[0033] Step 2: Dissolve Ammoniumpyrrolidinedithiocarbamate (PDTC) in normal saline. Take the animals from step 1 and give the model group PDTC intervention treatment every day, while the control group is injected with an equal volume of normal saline at the same time.

[0034] Step 3: Record the behavioral changes of the animals in step 2 daily; collect samples from the animals after the last administration to observe the pathological changes of brain tissue.

[0035] Preferably, the rodent in step 1 is a mouse.

[0036] Preferably, the PDTC is administered in step 2 by intraperitoneal injection.

[0037] Preferably, the dosage of PDTC in step 2 is 150 mg / kg / d.

[0038] Preferably, the animal behavioral tests in step 3 are seizure severity, latency, duration and survival rate.

[0039] Preferably, the animal brain tissue pathological detection in step 3 is hematoxylin-eosin staining or immunohistochemical staining.

[0040] The second aspect of the present invention provides an epilepsy animal model constructed by the above method.

[0041] The third aspect of the present invention provides the use of the above-mentioned epilepsy animal model in exploring the pathogenesis of epilepsy and evaluating anti-epileptic drugs.

[0042] In Examples 1 and 2 and Verification Examples 1 to 3 of the present invention, PDTC was purchased from Shanghai Biyuntian Biotechnology Co., Ltd., product number S1808. PDTC powder was diluted with sterile saline to a 30 mg / mL solution for immediate use. All experimental operators obtained a certificate of passing the Hubei Provincial Laboratory Animal Professional Technical Examination, and the injection protocol and procedures were approved by the Wuhan University Laboratory Animal Ethics Committee.

[0043] In Example 1, Example 2 and Effect Examples 1 to 3 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0044] The present invention will be further described below in conjunction with the embodiments:

[0045] Example 1: Method for constructing a drug-induced epilepsy model using PDTC

[0046] (1) Animal dosing and grouping

[0047] Normal male C57BL / 6 mice, 7-8 weeks old and weighing approximately 20 g, were purchased from the Experimental Animal Center of China Three Gorges University. All animals were housed under standard conditions with controlled temperature and adequate water and food. The mice were divided into a control group (n=5) and a model group (n=11). The model group received an intraperitoneal injection of 150 mg / kg PDTC using a 1 mL syringe, while the control group received an equal volume of normal saline.

[0048] (2) Observation of epileptic animal behavior

[0049] The seizure grade is based on the Racine grading system. Grade 0, no response; Grade 1, facial clonus; Grade 2, rhythmic nodding; Grade 3, one side of the forelimb is raised; Grade 4, both sides of the forelimb are raised, and the hind limbs stand; Grade 5, loss of balance, body rigidity, falling, jumping and running. The latent period is the interval from the start of drug administration to the first seizure to grade 3 or above. The duration is the continuous time of seizure grade 3 or above. Each animal was observed for 30 minutes after administration, and the results were recorded by two observers. The study found that when animals were given intraperitoneal injections of PDTC, the grade of epileptic seizures gradually increased with the increase in the number of injections (such as Figure 1 A and Table 1), the latency period of epileptic seizures gradually shortened (e.g. Figure 1 B and Table 2), the duration of epilepsy gradually prolonged (e.g. Figure 1 C and Table 3), while the survival rate began to decline after the fifth dose (as shown in Figure 1 D and Table 4).

[0050] Table 1 Seizure severity of each mouse after intraperitoneal injection of PDTC

[0051]

[0052] Table 1 corresponds to Figure 1 A's data.

[0053] Table 2. Latency of epilepsy seizures in mice after intraperitoneal injection of PDTC (s)

[0054]

[0055] Table 2 corresponds to Figure 1 B's data.

[0056] Table 3 Duration of epileptic seizures in mice after intraperitoneal injection of PDTC (s)

[0057]

[0058] Table 3 corresponds to Figure 1 C's data.

[0059] Table 4 Survival rate of mice after intraperitoneal injection of PDTC (%)

[0060] Number of PDTC injections 0 1 2 3 4 5 6 Survival rate 100 100 100 100 100 63.6 54.5

[0061] Table 4 corresponds to Figure 1 D's data.

[0062] Example 2 Brain tissue pathology detection

[0063] After the final intraperitoneal injection of PDTC in Example 1, the animals were anesthetized and positioned. Sterilized surgical instruments were used to incise the thorax, exposing the heart. 50 mL of normal saline was administered for apical perfusion. After the blood cells were completely perfused, 50 mL of 4% paraformaldehyde solution was administered for tissue fixation. Following perfusion, the brain tissue was removed and immersed in 4% paraformaldehyde solution at 4°C overnight. Dehydration was then performed using a gradient of alcohol, treated with xylene, and embedded in paraffin. 4 μm serial sections were cut using a microtome.

[0064] Effect Example 1 Observation of neuronal damage by hematoxylin-eosin staining

[0065] The paraffin sections obtained in Example 2 were fixed in an oven at 60°C for 2 hours, dewaxed with xylene, and then hydrated in sequence using 100% I, 100% II, 95%, 90%, 80%, and 70% alcohol. The membranes were broken with 0.3% Triton, washed with PBS, stained with eosin-hematoxylin solution, dehydrated in sequence with alcohol, and mounted with neutral gum after being transparentized with xylene.

[0066] The experimental results are as follows Figure 2 As shown in A and Table 5, hematoxylin-eosin staining studies found that neurons in the CA3 region of the hippocampus in the model group were condensed and the staining was deepened (e.g. Figure 2A), indicating that the PDTC-induced epilepsy model can cause neuronal damage in the hippocampal CA3 region (as shown in Table 5).

[0067] Table 5 Eosin-hematoxylin staining observation of the number of surviving neurons in each mouse after intraperitoneal injection of PDTC (units)

[0068]

[0069] Table 5 corresponds to Figure 2 A's data.

[0070] Effect Example 2: Immunohistochemical staining to observe glial cell activation

[0071] The paraffin sections obtained in Example 2 were fixed, dehydrated with alcohol in stages, and permeabilized with Triton. Endogenous catalase was inactivated using 3% hydrogen peroxide in methanol, and antigen retrieval was performed by microwave heating. Nonspecific sites were then blocked with 10% goat serum and incubated with primary antibodies against IBA1 (microglia marker) and GFAP (astroglial marker) at 4°C overnight. The next day, the sections were incubated with HRP secondary antibodies at room temperature, and developed using a DAB color development kit. The sections were washed with PBS, dehydrated in stages with alcohol, and transparentized with xylene before being mounted with neutral gum.

[0072] The experimental results are as follows Figure 2 B. As shown in Tables 6 and 7, IBA1 and GFAP immunohistochemical staining studies found that IBA1 (e.g. Figure 2 B and Table 6) and GFAP (as Figure 2 B and Table 7) showed no significant difference in expression compared with the control group, indicating that the PDTC-induced epilepsy model may not rely on inflammatory signals induced by microglia and astrocytes, which is different from the epilepsy models induced by other modeling drugs (such as kainic acid, ferric chloride, etc.).

[0073] Table 6 IBA1 immunohistochemical staining to observe microglial activation (%)

[0074]

[0075] Table 6 corresponds to Figure 2 B's data.

[0076] Table 7 GFAP immunohistochemical staining to observe astrocyte activation (%)

[0077]

[0078] Table 7 corresponds to Figure 2 B's data.

[0079] Effect Example 3: Comparison with other classic epilepsy models

[0080] An epilepsy model was established in C57BL / 6 mice (n=10) by intraperitoneal injection of 30 mg / kg of kainic acid (KA). KA was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. under the brand name K0250. The working solution was prepared with sterile saline and used immediately.

[0081] C57BL / 6 mice (n=10) were intraperitoneally injected with 37.5 mg / kg pentylenetetrazol (PTZ) every two days for six consecutive doses to establish an epilepsy model. PTZ was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. under the brand name P6500. The working solution was prepared with sterile saline and used immediately.

[0082] As shown in Table 8, the PDTC epilepsy model constructed in Example 1 was compared with the classic KA and PTZ epilepsy models. The study found that the PDTC epilepsy animal model has a high epilepsy induction rate and a high cost-effectiveness advantage.

[0083] Table 8 Comparison of production costs and induction rates of various epilepsy models

[0084]

[0085] Note: PDTC – 1g / 145 yuan; KA – 10mg / 2319.73 yuan; PTZ – 25g / 1074.4 yuan.

[0086] Fisher's exact test was used to analyze the difference in induction rate. *Compared with the PTZ modeling method, P=0.0108; **Compared with the PTZ modeling method, P=0.0039.

[0087] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for constructing an epilepsy mouse model, characterized in that: The NF-κB inhibitor PDTC was administered to mice by intraperitoneal injection at a dose of 150 mg / kg / d.

2. The construction method according to claim 1, wherein The mice were housed in a standard environment with free access to water and food.

3. The application of NF-κB inhibitor PDTC in constructing an epilepsy mouse model, characterized in that: The method for constructing the epilepsy mouse model comprises: administering the NF-κB inhibitor PDTC to the mouse; the administration method comprises intraperitoneal injection; and the administration dose is 150 mg / kg / d.

4. Use of the epilepsy mouse model obtained by the construction method according to claim 1 or 2 in exploring the pathogenesis of epilepsy or evaluating anti-epileptic drugs.

5. Use of the epilepsy mouse model obtained by the construction method according to claim 1 or 2 in the preparation of drugs for preventing or treating epilepsy.

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