Establishment and intervention strategy of working memory impairment model caused by prefrontal lobe ischemia

Abstract

The invention belongs to the field of medicines and application thereof, and particularly relates to establishment of a working memory impairment model and evaluation of the treatment effect of butylphthalide on working memory impairment caused by prefrontal cortex ischemia. The model is an animal model for working memory impairment caused by prefrontal cortex (mPFC) ischemia caused by endothelin (ET-1), and an intervention measure is butylphthalide medicine. By detecting related indexes of T maze, Y maze, white matter integrity, oligodendroglia cells (OLs) and ferroptosis of a mouse, it is evaluated that a model of working memory impairment caused by mPFC ischemia caused by ET-1 is established, and after the mouse is treated with butylphthalide, the working memory impairment of the mouse is improved, the white matter integrity is enhanced, and the OLs ferroptosis condition is reduced.

Description

Technical Field

[0001] The invention belongs to the field of medicine and its application, and specifically relates to the establishment of a working memory damage model and the evaluation of the therapeutic effect of butylphthalide on working memory damage caused by prefrontal lobe ischemia. Background Art

[0002] Anterior cerebral artery stroke accounts for 4.4% of strokes, causing ischemic damage to the prefrontal cortex (PFC), which seriously affects executive function, behavioral flexibility and working memory (WM). Oligodendrocytes (OLs) are very sensitive to ischemic damage. Ischemic stroke can lead to demyelination and cause working memory damage, but the specific mechanism is still unclear.

[0003] Studies have shown that iron-dependent phospholipid peroxidation and subsequent cell membrane damage induce ferroptosis during hemorrhagic stroke and multiple sclerosis, mediating the loss of OLs or oligodendrocyte precursor cells (OPCs) and demyelination. However, whether ferroptosis contributes to OLs loss and WM defects after PFC ischemia is unclear.

[0004] NBP is a drug approved by the National Medical Products Administration (NMPA) of China for the treatment of acute ischemic stroke. It can regulate the solute carrier family 7 (SLC7A11) / glutathione (GSH) / glutathione peroxidase 4 (GPX4) pathway and improve cognitive dysfunction by inhibiting ferroptosis in Parkinson's model mice. It is still unclear whether NBP can reduce ferroptosis in OLs after ischemia, thereby alleviating WM damage. Summary of the invention

[0005] The purpose of the present invention is to propose a method for constructing an animal model of working memory impairment and to evaluate the therapeutic effect of butylphthalide on working memory impairment caused by prefrontal ischemia.

[0006] The method for constructing an animal model of working memory impairment proposed in the present invention is to induce memory impairment in mice by microinjecting endothelin-1 (ET-1) into the prefrontal cortex region of mice, and to detect the therapeutic effect of butylphthalide on memory impairment caused by ischemia of the prefrontal cortex.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] In some specific embodiments of the present invention, the injection dose of ET-1 is 400 pmol / μL / side / mouse, and the administration dose of NBP is 30 mg / kg / d for 7 consecutive days.

[0009] The present invention provides application of mPFC ischemia caused by ET-1 in the process of damaging working memory of mice.

[0010] The present invention also provides the use of mPFC ischemia caused by ET-1 in causing damage to the integrity of mouse brain white matter and loss of oligodendrocytes.

[0011] The present invention also provides the use of mPFC ischemia caused by ET-1 in oligodendrocyte ferroptosis.

[0012] The present invention also provides the use of NBP in reducing the loss of oligodendrocytes in mPFC ischemia caused by ET-1.

[0013] The present invention also provides the use of NBP in alleviating brain white matter damage and protecting white matter integrity in mPFC ischemia caused by ET-1.

[0014] The present invention also provides the use of NBP in alleviating oligodendrocyte ferroptosis in mPFC ischemia caused by ET-1.

[0015] The experimental results of the present invention show that:

[0016] (1) ET-1-induced mPFC ischemia impairs working memory in mice.

[0017] (2) ET-1-induced mPFC ischemia leads to impaired white matter integrity and loss of OLs in mice.

[0018] (3) ET-1-induced mPFC ischemia induces OLs ferroptosis.

[0019] (4) NBP alleviates white matter damage during ET-1-induced mPFC ischemia and protects white matter integrity and OLs loss.

[0020] (5) NBP alleviates OLs ferroptosis during ET-1-induced mPFC ischemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0022] Figure 1Figure 2 shows the damage of mPFC ischemia caused by ET-1 on working memory in mice. (A) Experimental flow chart; (B) mPFC ischemia caused by ET-1 injection 0.5h later resulted in decreased cerebral blood flow (CBF) (n=4); (C) TTC-stained brain sections showed infarction caused by mPFC ischemia (n=4); (D) Nissl staining of brain sections showed mPFC ischemic damage (n=3); (EG) T-maze test showed that ET-1 treatment significantly reduced the percentage of correct choices (E) and upregulated Win-shift failure (F) and Lose-shift failure (G) (n=10); (H) Y-maze test ET-1 treatment significantly reduced the percentage of spontaneous alternations in the Y-maze task (n=10). **P<0.01 compared with the saline group.

[0023] Figure 2 The results showed that ET-1-induced mPFC ischemia resulted in impaired white matter integrity and loss of OLs in mice. (A) Immunofluorescence showed that the MBP level was significantly downregulated after mPFC ischemia in mice (n=6, scale bar=50μm); (B) Immunoblotting and quantitative results showed that ET-1 treatment significantly reduced the expression of MBP in mPFC in mice (n=7); (C) Immunofluorescence showed that the number of oligodendrocytes was significantly reduced after mPFC ischemia in mice (n=6, scale bar=50μm). Compared with the saline group,

[0024] **P<0.01.

[0025] Figure 3 Figure ET-1 induces ferroptosis of OLs caused by mPFC ischemia in mice. (A) Immunofluorescence showed that GPX4 expression in oligodendrocytes was reduced after mPFC ischemia (n=6, scale bar=50um); (B) Immunofluorescence showed that ferritin expression in oligodendrocytes was reduced after mPFC ischemia (n=6, scale bar=50um); (C) Immunoblotting showed that ET-1 treatment significantly reduced the expression of GPX4 and ferritin in the mPFC of mice (n=6); (D-E) Statistical graph of C; (F) ELISA detected that mPFC ischemia caused upregulation of free iron (n=7); (G) ELISA detected that mPFC ischemia caused upregulation of MDA (n=5); (H) ELISA detected that mPFC ischemia caused upregulation of LPO (n=6); (I) ELISA detected that mPFC ischemia caused downregulation of GSH (n=5). Compared with the saline group, *P<0.05, **P<0.01.

[0026] Figure 4NBP alleviates brain white matter damage during ET-1-induced mPFC ischemia, protects white matter integrity and OLs loss. (A) Schematic diagram of the experimental process; (B) NBP treatment significantly improved the ability to make correct choices in the T-maze induced by ET-1; (C) Effects of NBP treatment on the Win-shift in the T-maze; (D) NBP treatment improved the Lose-shift in the T-maze induced by ET-1 (n=9); (E) NBP treatment increased the percentage of spontaneous alternations in the Y-maze induced by ET-1 (n=9); (F) Immunofluorescence showed that NBP treatment promoted the expression of MBP induced by mPFC ischemia; (G) Western blot detection showed that NBP treatment significantly inhibited the decrease of MBP induced by mPFC ischemia (n=9); (H) Immunofluorescence detection showed that NBP treatment significantly inhibited the loss of oligodendrocytes induced by mPFC ischemia (n=6, scale=50μm). *P<0.05, **P<0.01.

[0027] Figure 5 Figure 3 shows that NBP alleviates OLs ferroptosis during ET-1-induced mPFC ischemia. (A) Immunofluorescence showed that NBP treatment significantly enhanced GPX4 expression in OLs induced by ET-1 (n=6, scale=50μm); (B) Immunofluorescence showed that NBP treatment significantly enhanced ferritin expression in OLs induced by ET-1 (n=6, scale=50μm); (C) Western blot showed that NBP treatment promoted the expression of GPX4 and ferritin induced by mPFC ischemia (n=3); (D-E) Statistical graph of C; (F) ELISA showed that NBP treatment (F) ELISA showed that NBP treatment significantly inhibited mPFC ischemia-induced free iron (n=6); (G) ELISA showed that NBP treatment significantly inhibited mPFC ischemia-induced MDA (n=4); (H) ELISA showed that NBP treatment significantly inhibited mPFC ischemia-induced LPO (n=6); (I) ELISA showed that NBP treatment significantly inhibited mPFC ischemia-induced GSH (n=8). Compared with the Vehicle group, *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0028] The present invention discloses a drug and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0029] The medicine provided by the present invention and the raw materials and reagents used in its application can all be purchased from the market.

[0030] The present invention will be further described below in conjunction with embodiments:

[0031] Example 1

[0032] 1. Male C57BL / 6 mice (weight 22-26g) were used to construct an ET-1-induced mPFC ischemia model. After surgery, mice were intraperitoneally injected with NBP / normal saline at 30 mg / kg / d for 7 consecutive days. The experiment was divided into three groups: (1) normal saline group; (2) ET-1 group; (3) ET-1+NBP group. On the 7th day after surgery, working memory was tested by T-maze and Y-maze tasks, infarction was tested by cerebral blood flow, TTC staining and Nissl staining, white matter integrity and OLs were tested by MBP and CC1, and OLs iron death was tested by free iron, MDA, LPO, and GSH.

[0033] 2. Methods

[0034] 2.1 Experimental design

[0035] The establishment of an animal model of working memory impairment caused by prefrontal ischemia was evaluated by using ET-1 to induce mPFC ischemia model, and by testing T-maze and Y-maze tasks, white matter integrity, and OLs loss. The ET-1 model group was treated with NBP to study the effect of NBP on alleviating white matter damage and improving working memory by inhibiting OLs ferroptosis.

[0036] 2.2 Experimental Animal Grouping

[0037] The experiment was divided into two groups: (1) saline group; (2) model group: ET-1; (3) treatment group: NBP.

[0038] 2.3 Establishment of mouse prefrontal cortex ischemia model

[0039] Procedure: Mice were anesthetized with isoflurane and the body temperature was maintained at 37°C with a heating pad during anesthesia. ET-1 was dissolved in saline to 400 pmol / μl. ET-1 (1 μl / side) or saline was injected into the mPFC at a rate of 0.4 μl / min (AP: 1.5, ML: ±0.4, DV: -2.5). After the injection was completed, the needle was left in place for 5 minutes.

[0040] 2.4 Catheterization and NBP administration

[0041] After mice were anesthetized with isoflurane, 23-gauge stainless steel catheters were inserted bilaterally. The mPFC coordinates were (AP: 1.5, ML: ±0.4, DV: -2.5). The guide cannula was fixed to the mouse skull using dental cement and sterile stainless steel screws. A sterile sham cannula was inserted into the cannula, and the mice were given a 7-day recovery period before the ET-1 modeling experiment was performed.

[0042] Two hours after ET-1 injection, mice were intraperitoneally injected with NBP / normal saline at 30 mg / kg / d for 7 consecutive days.

[0043] 2.5T Maze

[0044] The mice were fed a controlled diet and maintained at 85% of their initial body weight. They were allowed to drink water freely during the period. First, there was a 3-day adaptation period. The mice explored the T maze freely for 5 minutes every day. Food was placed in both target arms and replenished in time. Next was the training period, which was divided into 1 forced training and 9 formal training. During the forced training, food was placed at the ends of both arms. When the mice reached the end of any arm and received a food reward, they were immediately guided back to the trunk arm and the door was closed for 10 seconds for formal training. In the formal training, both target arms were open. The mice were counted as correct only when they chose the opposite arm and received a food reward. If the mice entered an arm they had entered before, it was counted as an incorrect choice and no reward was given. The interval between each training was 10 seconds, and 75% alcohol was used to remove the odor in the maze. The correct rate of the mice was counted every day. When the correct rate of the mice reached 70% for 3 consecutive days, the training was qualified and the training period ended. After that, there was a test period. Each mouse received 10 trials, and the number of errors of each mouse was recorded. The incorrect choice after the previous correct choice was recorded as Win-shift failure. An incorrect choice following the previous incorrect choice was recorded as a loss-shift failure. The total accuracy rate was calculated, and the higher the accuracy rate, the shorter the latency, indicating that the animal had better working memory.

[0045] 2.6Y Maze

[0046] The Y-shaped maze consists of three light-colored, opaque arms with an angle of 120°. Before the test, the mouse was placed in the center of the maze, and then the doors of the three arms were opened, allowing the mouse to freely explore the maze for 5 minutes. The test was performed 2 hours later. At the beginning of the test, the mouse was placed in the center of the maze, all doors were opened, and the mouse was free to explore the maze for 8 minutes. The order in which the mouse entered the arm was recorded. When the mouse entered different arms three times in a row, it was defined as a spontaneous alternation. The percentage of spontaneous alternation = number of spontaneous alternations / (total number-2)×100%.

[0047] 2.7TTC staining:

[0048] 24 hours after surgery, mice were anesthetized and perfused with cold PBS, and the brains were cut into 1 mm coronal slices. The brain slices were immersed in PBS containing 2% TTC at 37°C for 20 minutes, and then photographed and analyzed using Image J.

[0049] 2.8 Cerebral blood flow detection:

[0050] After anesthetizing the mice, the skull skin was separated along the midline incision, and RFLSIPro and laser speckle detection were used. The detection time was divided into before surgery, 0.5h, and 10h after surgery.

[0051] 2.9Western blot:

[0052] Mouse brain tissue and oligodendrocytes were lysed with cold RIPA lysis buffer and 10% protease inhibitor for 1 hour, and then centrifuged at 14000g for 15 minutes at 4°C. Protein concentration was determined using BCA protein detection kit.

[0053] Equal amounts of protein samples (30 μg) from each group were added to 12% SDS-PAGE gel for electrophoresis and then transferred to PVDF membrane. Blocked with 5% skim milk. Incubated with corresponding primary antibodies against MBP, GPX4, ferritin, and GADPH at 4°C overnight. After washing with TBST, incubated with enzyme-labeled anti-rabbit IgG or enzyme-labeled anti-mouse IgG at room temperature for 1 hour. Developed with enhanced luminescence solution and photographed.

[0054] 2.10 Immunofluorescence:

[0055] Mice were anesthetized with isoflurane and perfused with PBS and then 4% paraformaldehyde. Mouse brains were cut into 20 μm thick sections using a freezing microtome. The sections were blocked with 10% goat serum and 0.3% Triton X-100 for 2 hours at room temperature, and the sections were incubated with primary antibodies against MBP, GPX4, ferritin, and CC1 at 4°C overnight. Photos were taken after incubation with secondary antibodies at room temperature for 2 hours.

[0056] 2.11 Determination of malondialdehyde (MDA) and lipid peroxide (LPO)

[0057] Mouse brain tissue and oligodendrocytes were homogenized with PBS and the supernatant was collected after centrifugation. MDA and LPO levels in the lysate were detected using an MDA detection kit and a lipid peroxidation detection kit.

[0058] 2.12 Statistical analysis

[0059] Image J software was used to collect infarction photos and Western blot images, and GraphPad Prism statistical software was used for graphical analysis. The experimental data were expressed as mean ± standard deviation and analyzed statistically using SPSS13.0. P < 0.05 indicated statistical significance.

[0060] result:

[0061] Table 1 Correct choice rate in T maze after ET-1 injection in mPFC of mice

[0062]

[0063]

[0064] Table 2 Number of Win-shift failures in the T maze after ET-1 injection in the mPFC of mice

[0065]

[0066]

[0067] Table 3 Number of T-maze Lose-shift failures after ET-1 injection in the mPFC of mice

[0068]

[0069]

[0070] Table 4 Spontaneous alternation rate in the Y-maze after ET-1 injection in the mPFC of mice

[0071]

[0072]

[0073] Table 5 Western blot detection of MBP protein expression in PFC of mice after ET-1 injection

[0074]

[0075]

[0076] Table 6 Western blot detection of GPX4 protein expression in mPFC of mice treated with ET-1

[0077]

[0078]

[0079] Table 7 Western blot detection of ferritin expression in mPFC of mice treated with ET-1

[0080]

[0081]

[0082] Table 8 ELISA detection of free iron in mPFC of mice treated with ET-1

[0083]

[0084]

[0085] Table 9 ELISA detection of MDA in mPFC of mice treated with ET-1

[0086]

[0087]

[0088] Table 10 ELISA detection of LPO in mice after ET-1 treatment in mPFC

[0089]

[0090]

[0091] Table 11 ELISA detection of GSH in mPFC of mice treated with ET-1

[0092]

[0093]

[0094] Table 12 Correct choice rate of NBP-treated mPFC ischemic mice in T-maze test

[0095]

[0096]

[0097] Table 13 Win-shift failure times in NBP-treated mPFC ischemic mice tested in T maze

[0098]

[0099]

[0100] Table 14 T-maze test of the number of Lose-shift failures in NBP-treated mPFC ischemic mice

[0101]

[0102]

[0103] Table 15 Spontaneous alternation rate in NBP-treated mPFC ischemic mice tested in Y-maze

[0104]

[0105]

[0106] Table 16 Western blot detection of MBP protein expression in NBP-treated mPFC ischemic mice

[0107]

[0108]

[0109] Table 17 Western blot detection of GPX4 protein expression in mPFC of mice treated with NBP

[0110]

[0111]

[0112] Table 18 Western blot detection of ferritin expression in mPFC of mice treated with NBP

[0113]

[0114]

[0115] Table 19 ELISA detection of free iron expression in mPFC of mice treated with NBP

[0116]

[0117]

[0118] Table 20 ELISA detection of MDA expression in mPFC of mice treated with NBP

[0119]

[0120]

[0121] Table 21 ELISA detection of LPO expression in mPFC of mice treated with NBP

[0122]

[0123]

[0124] Table 22 ELISA detection of GSH expression in mPFC of mice treated with NBP

[0125]

[0126]

Claims

1. An animal model, characterized in that: Ischemia induced by ET-1 injection into the prefrontal cortex of mice can be used to detect and evaluate working memory impairment.

2. Use of the animal model according to claim 1 in preparing an animal model of working memory impairment in mice.

3. Use of the animal model according to claim 1 in preparing an animal model of impaired white matter integrity and OLs loss in mice.

4. Use of the animal model according to claim 1 in preparing an animal model of ferroptosis in mouse OLs.

5. A drug, characterized in that 3-n-Butylphthalide (NBP) is a drug approved by the China National Medical Products Administration (NMPA) for the treatment of acute ischemic stroke.

6. The drug according to claim 5, characterized in that The effective dose of NBP was 400 pmol / μL / side / animal.

7. Use of the drug according to claim 5 in the preparation of a drug for alleviating white matter damage after mPFC ischemia induced by ET-1.

8. Use of the drug according to claim 5 in the preparation of a drug for protecting white matter integrity after ET-1-induced mPFC ischemia.

9. Use of the drug according to claim 5 in preparing a drug for reducing OLs loss after mPFC ischemia induced by ET-1.

10. Use of the drug according to claim 5 in the preparation of a drug for alleviating OLs ferroptosis after mPFC ischemia induced by ET-1.

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