Construction method of photochemically induced mouse cerebral ischemia model

Abstract

The invention belongs to the field of biological medicines, and provides a construction method of a photochemically induced mouse cerebral ischemia model, which comprises the following steps: S1, inducing anesthesia of a mouse, and ensuring that the mouse is continuously in an anesthesia state; s2, in the lateral position of the mouse, unhairing and disinfecting the face on one side, and cutting from the face of the mouse to expose the proximal end position of the M3 segment of the middle cerebral artery of the mouse or the cross point of reverse extension lines of branch blood vessels of the middle cerebral artery; s3, injecting a photosensitizer into the mouse, and after the photosensitizer circulates uniformly in the body of the mouse, irradiating the proximal end of the M3 segment of the middle cerebral artery or the cross point of the reverse extension line of the branch blood vessel of the middle cerebral artery after laser focusing; s4, after laser irradiation is finished, the skin is sutured, anesthesia is stopped, and the wound of the mouse is coated with an analgesic. The model obtained by the construction method of the photochemically induced mouse cerebral ischemia model provided by the invention has a larger infarct volume, and the infarct invades the subcortical brain region, so that the pathological characteristics of ischemic brain tissues are more obvious.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and particularly relates to a method for constructing a photochemically induced mouse cerebral ischemia model. Background Art

[0002] Stroke, also known as apoplexy or cerebrovascular accident, is a common and frequently occurring disease that seriously threatens human health. Stroke is divided into ischemic and hemorrhagic types, of which ischemic stroke is the most common type of stroke in clinical practice (accounting for about 80% of the incidence rate). It is mainly caused by various emboli in the blood (such as mural thrombi in the heart, plaques of atherosclerosis, etc.) entering the cerebral artery with the blood flow and blocking the blood vessels, causing ischemic necrosis of brain tissue in the artery blood supply area, and then focal neurological deficits. 2 .

[0003] Currently, the main treatment for ischemic stroke is drug thrombolysis. Recombinant tissue-type plasminogen activator (rtPA) is the only FDA-approved thrombolytic drug for ischemic stroke, but its effective treatment window is narrow, only 3.5-4.5 hours. Using thrombolytic drugs beyond this time is very likely to cause cerebral hemorrhage and endanger life. 3 .

[0004] The main research method at present is to simulate the characteristics of stroke through animal models to develop therapeutic drugs or study the pathogenesis. The most commonly used animal model of stroke is to use thread embolism to occlude the middle cerebral artery (MCAO). The ischemic stroke infarction volume formed by this method is stable, and the animals show obvious neurological dysfunction, but this model does not involve the formation of thrombus, so it is not suitable for drug thrombolysis-related research.

[0005] There are very limited types of preclinical animal models suitable for drug thrombolysis research. The most commonly used modeling method is to use autologous blood to create thrombi in vitro and then re-inject them into the middle cerebral artery of mice through a catheter. However, this method requires extremely high surgical skills, complicated steps, and a low success rate for modeling. 4 In addition to the above methods, there is also a method of creating a thrombus by applying low-frequency direct current to the common carotid artery, and then mechanically crushing the thrombus so that it enters the blood supply area of ​​the middle artery under the impact of blood flow to achieve infarction. This method still requires high surgical skills, and it is impossible to accurately control the location of the thrombus blockage, so the stability of the model is relatively poor. 5 .

[0006] The photochemically induced mouse cerebral ischemia model is to inject photosensitive substances intravenously and use them to generate free radicals under green laser, directly damaging vascular endothelial cells, causing them to produce lipid peroxides, promoting platelet aggregation, activating the coagulation process, forming thrombi in situ at the site of vascular damage, blocking blood vessels and causing ischemia. This model was first created by Watson et al. in 1985. 6 . This model is less traumatic to experimental animals and has a high success rate in modeling, so it is widely used in permanent cerebral infarction and related research. However, since its inception, the model has been aimed at blocking the blood supply area of ​​the middle cerebral artery (MCA), and has not considered whether drugs such as rtPA can be used for research on cerebral ischemia-reperfusion injury and thrombolytic drugs. In addition, this model uses scattered laser to irradiate the surface of the skull, and the infarction formed is limited to the superficial surface of the cerebral cortex at the irradiation location. Therefore, the neurological function damage of this type of model is very mild, and common neurological function scores cannot be used to evaluate the neurological function of animals. Therefore, further developing and improving existing preclinical animal models of stroke is of great significance for the treatment and research of stroke.

[0007] In the following decades after the establishment of the photochemically induced stroke animal model, a number of improved methods based on this model have emerged. 7 In 2004, et al. made a surgical incision in the midline between the mouse eye and the inner edge of the ear. By using a convex lens to focus the laser irradiation closer to the proximal end of the MCA, combined with ligation of the ipsilateral common carotid artery, they created a light-induced middle cerebral artery embolism model with a more stable infarct volume. However, the infarct volume of this model is still small, and the infarct location is almost entirely concentrated in the cortical area, failing to affect brain areas such as the hippocampus, and neurological function damage is not obvious. In addition, this study has not yet conducted research on cerebral ischemia-reperfusion injury and thrombolytic drugs using drugs such as rtPA.

[0008] Sun 8 et al. found in 2020 that the injection of rtPA in the above-mentioned model did not play a good thrombolytic effect, and the perfusion level was very low. Therefore, the method of injecting thrombin at the same time as injecting the photosensitizer-Bengal rose was adopted, combined with low-power, long-term laser irradiation to create a light-induced thrombus that is more sensitive to rtPA. The light-induced stroke model created by this method can be used for thrombolytic research, but the common carotid artery needs to be ligated during modeling, and the infarction volume is still very small (less than 20%), and the infarction location is still mainly concentrated in the cortical area, and the neurological damage is not obvious, so it still has great limitations in application. Summary of the invention

[0009] In order to solve the problems in the prior art that the animal stroke model produced by the photochemical induction method has a small infarct volume, no obvious neurological deficit, and poor response to thrombolytic drugs, the present invention provides a method for constructing a photochemically induced mouse cerebral ischemia model.

[0010] The method for constructing the photochemically induced mouse cerebral ischemia model comprises the following steps:

[0011] S1: Induce anesthesia in mice and ensure that the mice remain in anesthesia, monitor the rectal temperature of the mice and heat to maintain 37±0.5°C;

[0012] S2: The mouse is placed in a lateral position, one side of the face is depilated and disinfected, and an incision is made on the face of the mouse to expose the proximal end of the M3 segment of the middle cerebral artery of the mouse, or the intersection of the reverse extension lines of the branch vessels of the middle cerebral artery;

[0013] S3: injecting a photosensitizer into the mouse, and after the photosensitizer circulates evenly in the mouse, focusing the laser to irradiate the proximal end of the M3 segment of the middle cerebral artery or the intersection of the reverse extension lines of the middle cerebral artery branch vessels;

[0014] S4: After the laser irradiation, the skin was sutured, anesthesia was stopped, and analgesics were applied to the wounds of the mice.

[0015] Further, S2 comprises the following steps:

[0016] A first incision was made from the outer corner of the mouse eye to the inner edge of the ear, the skin was bluntly separated until the boundary between the edge of the skull and the facial skeletal muscle was visible, the muscles were cut along the boundary of the skull head and face using microscissors, a second incision was made to expose the maxilla under the muscle, a third incision was made vertically along the midline of the second incision to separate the upper and lower muscles, fully expose the maxilla and temporomandibular joint, and confirm the proximal end position of the middle cerebral artery M3 segment, or the intersection of the reverse extension line of the middle cerebral artery branch vessels.

[0017] Furthermore, the length of the second incision is 0.3 cm.

[0018] Furthermore, the photosensitizer in step S3 is rose bengal, and the dosage is 50 mg / kg.

[0019] Furthermore, the wavelength of the irradiated laser was 520 nm, the power was 40 mW, and the irradiation time was 3 minutes.

[0020] Furthermore, the laser spot diameter is 0.7 mm.

[0021] Furthermore, during the laser focusing process, a laser with a frequency of 10-20 mW can be used to adjust the spot size and irradiation position.

[0022] Beneficial effects:

[0023] 1. The infarct volume of the model obtained by the method for constructing the photochemically induced mouse cerebral ischemia model provided by the present invention is larger, and the infarction invades the subcortical brain area, so the pathological characteristics of the ischemic brain tissue are more obvious.

[0024] 2 The neurological dysfunction of mice caused by the photochemically induced mouse cerebral ischemia model construction method provided by the present invention is more obvious, which is suitable for research related to neurological function and motor function.

[0025] 3. The method for constructing the photochemically induced mouse cerebral ischemia model provided by the present invention is less invasive and only requires a facial incision, without a neck incision or ligation of the common carotid artery. Therefore, the ischemia of the brain tissue is completely caused by thrombosis, which is more similar to the clinical situation.

[0026] 4. The model obtained by the method for constructing the photochemically induced mouse cerebral ischemia model provided by the present invention can achieve thrombolysis without the need for additional injection of other reagents, and is therefore more similar to the clinical situation and the model operation is also simpler. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A flow chart of the method for constructing a photochemically induced mouse cerebral ischemia model provided by the present invention;

[0028] Figure 2 A schematic diagram of the first incision position provided in Example 1;

[0029] Figure 3 A schematic diagram of the positions of the second incision and the third incision provided in Example 1;

[0030] Figure 4 A schematic diagram of the intersection position of the reverse extension lines of the middle cerebral artery branch vessels provided in Example 1;

[0031] Figure 5 Schematic diagram of the laser provided in Example 1 focusing on the proximal end of the M3 segment of the middle cerebral artery;

[0032] Figure 6 A schematic diagram of irradiating the proximal end of the M3 segment of the middle cerebral artery with laser light having a wavelength of 520 nm and a power of 40 mW provided in Example 1;

[0033] Figure 7 A schematic diagram of blood flow in the branches of the middle cerebral artery after laser irradiation provided in Example 1;

[0034] Figure 8 Laser speckle blood flow imaging of the modeling site before and after laser irradiation provided in Example 1;

[0035] Fig. 9The mouse brain magnetic resonance imaging provided in Example 1;

[0036] Fig.10 This is a TTC staining image of the brain taken 48 hours after the modeling provided in Example 1;

[0037] Fig.11 Laser speckle blood flow imaging provided for Application Example 1. DETAILED DESCRIPTION

[0038] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention are described in detail below with reference to the accompanying drawings, but they should not be construed as limiting the applicable scope of the present invention.

[0039] The present invention provides a method for constructing a photochemically induced mouse cerebral ischemia model. The present invention improves the irradiation position, power and irradiation time of the focused laser, selects a position as close as possible to the proximal end of the MCA, and performs high-power, short-time irradiation. The model constructed by the method for constructing the photochemically induced mouse cerebral ischemia model provided by the present invention is less invasive, can cause stable cerebral infarction without ligating the common carotid artery, and the infarction volume is larger, is not limited to the cortical area of ​​the brain surface, and can affect the functions of more brain regions, thereby showing obvious neurological damage. In addition, the method for constructing the photochemically induced mouse cerebral ischemia model provided by the present invention adopts high-power, short-time focused irradiation of the MCA, and the spot influence area is small, so the formed thrombus responds well to rtPA. After intravenous injection of rtPA, the blood supply to the ischemic area is significantly restored, so it is suitable for related research on thrombolytic drugs and ischemia-reperfusion injury.

[0040] See also Figure 1 The method for constructing a photochemically induced mouse cerebral ischemia model provided by the present invention comprises the following steps:

[0041] S1: Induce anesthesia in mice and ensure that the mice remain in anesthesia, monitor the rectal temperature of the mice and heat to maintain 37±0.5°C;

[0042] S2: The mouse is placed in a lateral position, one side of the face is depilated and disinfected, and an incision is made on the face of the mouse to expose the proximal end of the M3 segment of the middle cerebral artery of the mouse, or the intersection of the reverse extension lines of the branch vessels of the middle cerebral artery;

[0043] S3: injecting a photosensitizer into the mouse, and after the photosensitizer circulates evenly in the mouse, focusing the laser to irradiate the proximal end of the M3 segment of the middle cerebral artery or the intersection of the reverse extension lines of the middle cerebral artery branch vessels;

[0044] S4: After the laser irradiation, the skin was sutured, anesthesia was stopped, and analgesics were applied to the wounds of the mice.

[0045] During the laser focusing process, a 10-20mW laser can be used to adjust the spot size and irradiation position. After the laser spot size and irradiation position are determined, a 40mW laser is used to irradiate and coagulate blood at the proximal end of the M3 segment of the middle cerebral artery or at the intersection of the reverse extension line of the middle cerebral artery branch vessels to construct a mouse cerebral ischemia model.

[0046] According to the above-mentioned method for constructing a photochemically induced mouse cerebral ischemia model, Example 1 is provided.

[0047] Example 1

[0048] See also Figure 2-11 The method for constructing a photochemically induced mouse cerebral ischemia model comprises the following steps:

[0049] 1. Six mice were modeled, and anesthesia was induced in the mice using a 3% isoflurane-air mixture (the volume fraction of isoflurane in the air mixture was 3%) through a mouse breathing mask; 1.0-1.5% isoflurane-air mixture (the volume fraction of isoflurane in the air mixture was 1.0-1.5%) was used to maintain the mice in an anesthetized state during surgery. During the operation, the body temperature of the mice was monitored by rectal temperature and maintained at 37±0.5℃ by a heating pad.

[0050] 2. Place the mouse in lateral decubitus position, use depilatory cream to remove hair on one side of its face, and use iodine tincture and 75% ethanol to wipe and disinfect the depilatory area.

[0051] 3. Such as Figure 2 As shown in the figure, the first incision was made from the outer corner of the mouse eye to the inner edge of the ear, and the skin was bluntly separated to reveal the boundary between the edge of the skull and the facial skeletal muscle. Figure 3 As shown, use microscissors to cut the muscles along the boundary of the skull head and face, make a second incision about 0.3 cm long, and expose the maxilla under the muscle. Make a third incision vertically along the midline of the second incision to separate the upper and lower muscles and fully expose the maxilla and temporomandibular joint.

[0052] 4. Such as Figure 4 As shown, the proximal end of the middle cerebral artery (MCA) M3 segment is identified. When the blood vessel cannot be clearly seen, the intersection of the reverse extension lines of its branch vessels (blue arrows) is regarded as the proximal end of the middle cerebral artery M3 segment.

[0053] 5. As Figure 5 , turn on the laser light source (Beijing Honglan Optoelectronics Technology Co., Ltd.), adjust the position of the low-power laser (20 mW) collimator head, aim it at the proximal end of the middle cerebral artery M3 segment, and focus the laser to minimize the laser spot diameter, about 0.7 mm.

[0054] 6. Inject 100 μl of photosensitizer, Red Bengal, into the mouse’s tail vein at a dose of 50 mg / kg. Wait for 5 minutes after injection to allow the photosensitizer to circulate evenly in the mouse’s body.

[0055] 7. Such as Figure 6 As shown, the proximal end of the M3 segment of the middle cerebral artery was irradiated with a laser having a wavelength of 520 nm and a power of 40 mW for 3 minutes.

[0056] 8. After irradiation, Figure 7 As shown, the blood flow in the branch vessels of the middle cerebral artery disappeared. The skin was sutured, the anesthetic gas supply was stopped to allow the mouse to wake up naturally, and lidocaine gel was applied to the wound for analgesia.

[0057] Step 7: Laser speckle blood flow imaging of the modeling site before and after laser irradiation and coagulation. The results are shown in Figure 8 By comparison, it can be seen that the arterial blood supply area is blocked.

[0058] After modeling, the mice were tested and rated as follows:

[0059] 1. Magnetic resonance imaging of the mouse brain was performed immediately after modeling, and then every few hours until 24 hours. The results were shown in Fig. 9 , from the figure, we can clearly see the process of infarction gradually invading the subcortical tissue.

[0060] 2. After 48 hours of modeling, the brain was taken for TTC staining, and the infarct volume of the models created by the suture tMCAO, light induction method and the method for constructing the photochemical induced mouse cerebral ischemia model provided by the present invention (improved light induction method) can be seen. The results are shown in Fig.10 .

[0061] 3. Model nerve injury rating, the rating method is as follows:

[0062] Mouse neurologic severity score was performed on the mice 1 hour after waking up. 9 The score includes movement, perception and conditioned reflex, with a total of 14 points. 1 point is given for failure to complete the target action. The final score is added up, 1-4 points for mild, 5-9 points for moderate, 10-14 points for severe neurological impairment, and the results are displayed in Fig.10 .

[0063] pass Fig.10It can be seen that the infarct volume of the suture-induced tMCAO was 31.64±4.57%, and the mouse nerve injury score was 9.50±2.58 points; the infarct volume of the light-induced ipMCAO model produced by the method provided was 22.31±2.23%, and the mouse nerve injury score was 7.33±1.03 points; while the infarct volume of the traditional light-induced pMCAO was 9.34±3.11%, and the mouse nerve injury score was only 2.00±0.63 points. The statistical method used one-way analysis of variance combined with Turkey test, *P<0.05, ns=not significance.

[0064] Application Example 1

[0065] Thrombolysis operation is performed on the model obtained by the method for constructing the photochemically induced mouse cerebral ischemia model provided by the present invention

[0066] When the model obtained by the construction method of the photochemically induced mouse cerebral ischemia model provided by the present invention is subjected to thrombolysis, the mouse is anesthetized again after a period of embolism, the neck is wiped and disinfected with iodine tincture and 75% ethanol, and a longitudinal incision of about 1 cm in length is made above the clavicle of the mouse 1 cm to the right of the anterior midline of the neck using ophthalmic scissors to expose the jugular vein, and rtPA (10 mg / kg) is injected intravenously, of which 10% is a push injection and 90% is completed within 30 minutes. After completion, press with sterile gauze for 1 minute to stop bleeding, suture the skin, and apply lidocaine gel to the wound for analgesia. After thrombolysis, the mouse can eat and drink freely. When sampling, the mouse is deeply anesthetized with isoflurane. Cardiac perfusion is performed using heparin-normal saline precooled at 4°C. After completion, the mouse is dislocated, and the brain is removed for laser speckle blood flow imaging, and the results are shown in Fig.11 .from Fig.11 It can be seen that 60 minutes after irradiation, blood flow decreased to 21.67±6.18% before irradiation. After thrombolysis, blood flow recovered to 58.67±4.70% before irradiation. The statistical method used two-way ANOVA combined with Holm-Sidak test, *P<0.05.

[0067] In this example, male C57BL / 6 mice were used, with no weight restriction.

[0068] References:

[0069] 1. Dong, Q., et al. The Chinese Stroke Association scientific statement: intravenous thrombolysis in acute ischaemic stroke. Stroke Vasc Neurol 2, 147-159 (2017).

[0070] 2.Hankey,G.J.Stroke.Lancet 389,641-654(2017).

[0071] 3.Emberson,J.,et al.Effect of treatment delay,age,and stroke severityon the effects of intravenous thrombolysis with alteplase for acute ischaemicstroke:a meta-analysis of individual patient data from randomisedtrials.Lancet 384,1929-1935(2014).

[0072] 4.Fluri,F.,Schuhmann,M.K.&Kleinschnitz,C.Animal models of ischemicstroke and their application in clinical research.Drug Des Devel Ther 9,3445-3454(2015).

[0073] 5.Ma,Y.,et al.Effect of recombinant plasminogen activator timing onthrombolysis in anovel rat embolic stroke model.Pharmacol Res 107,291-299(2016).

[0074] 6.Watson,B.D.,Dietrich,W.D.,Prado,R.&Ginsberg,M.D.Argon laser-inducedarterialphotothrombosis.Characterization and possible application to therapyof arteriovenousmalformations.J Neurosurg 66,748-754(1987).

[0075] 7.Sugimori,H.,Yao,H.,Ooboshi,H.,Ibayashi,S.&Iida,M.Krypton laser-inducedphotothrombotic distal middle cerebral artery occlusion withoutcraniectomy in mice.Brain ResBrain Res Protoc 13,189-196(2004).

[0076] 8.Sun,Y.Y.,et al.A murine photothrombotic stroke model with anincreased fibrin contentand improved responses to tPA-lytic treatment.BloodAdv 4,1222-1231(2020).

[0077] 9.Li,Y.,et al.Intrastriatal transplantation of bone marrownonhematopoietic cells improvesfunctional recovery after stroke in adultmice.J Cereb Blood Flow Metab 20,1311-1319(2000)。

Claims

1. A method for constructing a photochemically induced mouse cerebral ischemia model, characterized in that: The following steps are involved: S1: Induce anesthesia in mice and ensure that the mice remain in anesthesia, monitor the rectal temperature of the mice and heat to maintain 37±0.5°C; S2: The mouse is placed in a lateral position, one side of the face is depilated and disinfected, and an incision is made on the face of the mouse to expose the proximal end of the M3 segment of the middle cerebral artery of the mouse, or the intersection of the reverse extension lines of the branch vessels of the middle cerebral artery; S3: injecting a photosensitizer into the mouse, and after the photosensitizer circulates evenly in the mouse, focusing the laser to irradiate the proximal end of the M3 segment of the middle cerebral artery or the intersection of the reverse extension lines of the middle cerebral artery branch vessels; S4: After the laser irradiation, the skin was sutured, anesthesia was stopped, and analgesics were applied to the wounds of the mice.

2. The method for constructing a photochemically induced mouse cerebral ischemia model according to claim 1, characterized in that: S2 includes the following steps: A first incision was made from the outer corner of the mouse eye to the inner edge of the ear, the skin was bluntly separated until the boundary between the edge of the skull and the facial skeletal muscle was visible, the muscles were cut along the boundary of the skull head and face using microscissors, a second incision was made to expose the maxilla under the muscle, a third incision was made vertically along the midline of the second incision to separate the upper and lower muscles, fully expose the maxilla and temporomandibular joint, and confirm the proximal end position of the middle cerebral artery M3 segment, or the intersection of the reverse extension line of the middle cerebral artery branch vessels.

3. The method for constructing a photochemically induced mouse cerebral ischemia model according to claim 2, characterized in that: The length of the second incision is 0.3 cm.

4. The method for constructing a photochemically induced mouse cerebral ischemia model according to claim 1, characterized in that: The photosensitizer in step S3 is red bengal with a dosage of 50 mg / kg.

5. The method for constructing a photochemically induced mouse cerebral ischemia model according to claim 1, characterized in that: The wavelength of the irradiated laser was 520 nm, the power was 40 mW, and the irradiation time was 3 minutes.

6. The method for constructing a photochemically induced mouse cerebral ischemia model according to claim 1, characterized in that: The laser spot diameter is 0.7 mm.

7. The method for constructing a photochemically induced mouse cerebral ischemia model according to claim 1, characterized in that: During the laser focusing process, a 10-20mW frequency laser can be used to adjust the spot size and irradiation position.

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