Application of rat SAH model in cognitive function research

Abstract

The invention relates to the technical field of biomedicine, and discloses application of a rat SAH model in cognitive function research. The construction method of the rat SAH model comprises the following steps: step 1, marking a modeling position on the occipital neck part of a rat, and performing rat SAH modeling under ultrasonic guidance to obtain the rat SAH model; step 2, performing ultrasonic scanning on the rat SAH model and the false operation group constructed in the step 1, and verifying whether the rat SAH model is successfully modeled or not; step 3, performing magnetic resonance scanning on the rat SAH model and the false operation group constructed in the step 1, and verifying whether the cognitive function of the rat SAH model is affected or not; and step 4, observing rat cerebral nerve fibers of the rat SAH model and the false operation group constructed in the step 1. According to the application of the rat SAH model in cognitive function research, the constructed rat SAH model is good in stability, high in success rate and obvious in model behavior, and can be used for research of diseases such as cognitive impairment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of a rat SAH model in cognitive function research. Background Art

[0002] Subarachnoid hemorrhage (SAH) is an acute hemorrhagic cerebrovascular disease caused by rupture of intracranial blood vessels, which causes blood to flow into the subarachnoid space. 85% of cases are caused by rupture of intracranial aneurysms. Although medical diagnosis and treatment methods have made great progress, its mortality and disability rates are still very high. Poor prognosis is closely related to brain damage after SAH, including early brain injury (EBI) and delayed cerebral ischaemia (DCI). Therefore, it is of great clinical significance to explore the pathological mechanism of brain damage after SAH and seek relevant measures to improve prognosis.

[0003] The SAH model (subarachnoid hemorrhage model) is an important tool for studying the pathological mechanism and treatment methods of subarachnoid hemorrhage (SAH). SAH refers to the flow of blood into the subarachnoid space, which is the area between the arachnoid membrane and the pia mater surrounding the brain. It accounts for 5% to 10% of all strokes. Although its incidence is low, SAH poses a serious threat to human health due to its high mortality and disability rates.

[0004] The SAH model is widely used to study the pathological mechanism, biomarkers and treatment methods of SAH. For example, the SAH model can be used to study the expression of oxygen free radicals and intercellular adhesion molecule-1 (ICAM-1) and its relationship with cerebral vasospasm, so as to gain a deeper understanding of the pathogenesis of SAH. In addition, the SAH model is also used to evaluate the efficacy and safety of different treatment methods and provide a basis for clinical treatment. In short, the SAH model is of great significance in basic and clinical medical research. By continuously optimizing the model preparation method and application scenarios, we can better understand the pathological mechanism of SAH and explore effective treatment methods.

[0005] There are many methods for making rat SAH models. So far, the methods for making rat SAH models can be mainly summarized into three categories: (1) intracranial artery puncture method; (2) occipital cistern blood injection method; (3) prechiasmatic cistern or subarachnoid space blood injection method.

[0006] The first method is to puncture a certain point in the intracranial segment of the internal carotid artery, and blood flows into the subarachnoid space. This method is consistent with the pathogenesis of subarachnoid hemorrhage, but it has certain defects: ① After the fine line is inserted into the blood vessel, it may cause temporary cerebral ischemia. If the time exceeds 30 minutes, there is a possibility of local brain tissue necrosis; ② The amount of bleeding is not constant, which affects the observation of experimental results; ③ The operation is complicated, the damage is large, and the mortality rate is high. About 50% of the animals died 24 hours after subarachnoid hemorrhage; ④ It can cause intracerebral hemorrhage, and the incidence rate is about 11% of all experimental animals. In recent years, there have been many reports on the establishment of SAH models by craniotomy and blood injection in the optic chiasm cistern. This method uses a brain stereotaxic instrument to fix, and the coronal suture, sagittal suture and bregma point are exposed surgically. After drilling a hole 7.5mm in front of the bregma point and slightly to the right, the optic chiasm cistern is injected with blood. However, this method requires instrument assistance, and the operation process is complicated, and it requires special training to be proficient.

[0007] The latter two modeling methods are mostly surgical craniotomy to inject non-anticoagulated autologous blood into the subarachnoid space through the forehead or cerebellomedullary cistern or lateral ventricle cannula, but there are many methods that use autologous arterial blood to inject into the cerebellomedullary cistern, which is called the occipital cistern blood injection method. Studies have shown that SAH rat models require models to have similar pathogenesis, strong controllability of operation, the difference between all models should be as small as possible, and the mortality rate should not be too high. Therefore, the present invention provides a stable rat SAH model with a high success rate, and studies the application of the model in cognitive function. Summary of the invention

[0008] The purpose of the present invention is to provide an application of a rat SAH model in cognitive function research. The constructed rat SAH model has good stability, a high success rate, and obvious model behavior, and can be used for the research of diseases such as cognitive impairment.

[0009] To achieve the above object, the present invention provides an application of a rat SAH model in cognitive function research. The method for constructing a rat SAH model is as follows:

[0010] Step 1, selecting normal rats, marking the modeling position on the occipital and cervical regions of the rats, and performing SAH modeling on the rats under ultrasound guidance to obtain a rat SAH model;

[0011] Step 2, performing ultrasound scanning on the rat SAH model constructed in step 1 and the sham operation group to verify whether the rat SAH model is successfully established;

[0012] Step 3, performing magnetic resonance imaging on the rat SAH model constructed in step 1 and the sham operation group to verify whether the cognitive function of the rat SAH model is affected;

[0013] Step 4, observing the brain nerve fibers of the rats in the SAH model and sham operation group constructed in step 1.

[0014] Furthermore, in step 1, when performing the SAH modeling in rats under ultrasound guidance, a high-frequency ultrasound probe is used, and the frequency of the ultrasound probe is 10-12 MHz.

[0015] Further, the detailed steps of step 1 are as follows:

[0016] The rat was placed in a prone position on a foam pad, with the head naturally hanging down over the edge of the pad and resting on the table to form a 45°-60° tilt angle. First, the ultrasound probe was placed at the cistern magna, keeping the needle at an upward angle of 60-80° to find the cistern, and then a 1 mm inner diameter limiting sleeve was placed on the puncture needle to ensure that only a 4 mm length of the needle tip could penetrate the skin. The 4 mm length can prevent the needle from entering too shallowly or too deeply, effectively improving the success rate of the model.

[0017] Fix the skin of the rat's head and neck, use a 26G puncture needle to puncture from the marked puncture point at an angle parallel to the head and aligned with the line from the external auditory canal. At the same time, observe whether there is a high echo position in the cistern of the occipital magna through ultrasound, then advance the needle in a small amplitude, and inject a small dose of blood to observe whether there is blood in the cistern of the occipital magna. If blood is injected, it indicates that the position is correct and the rat SAH model is successfully established.

[0018] Furthermore, the magnetic resonance scan in step 3 was completed using a fast echo DTI sequence scan with the following scanning parameters: TR = 2605 ms, TE = 30.85 ms, four segments, b = 1000 s / mm 2 , 30diffusion directions, five B0images, matrix=128×128pixels2, FOV=30×30mm 2 , slice thickness = 2.0 mm;

[0019] T2Flair sequence: TE = 6000ms, TR = 102ms, Matrix = 256 × 256 × 128 pixels3,

[0020] FOV = 20 × 20 × 20 mm 3 ; slice thickness = 2.0 mm;

[0021] DWI sequence: TE = 3990 ms, TR = 56 ms, Matrix = 256 × 256 × 128 pixels3, FOV = 20 × 10 × 20 mm 3 ;slice thickness=2.0mm.

[0022] Further, it is applied to cognitive function research. The advantages and positive effects of the application of the rat SAH model described in the present invention in cognitive function research are:

[0023] 1. The present invention performs SAH modeling in rats under ultrasound guidance, using a high-frequency linear ultrasound probe with a frequency of 10 to 12 MHz. The high-frequency probe has a very high resolution, which helps to correctly identify the location of the cistern magnum, so that the location of the needle can be found in time during modeling, preventing the needle from being inserted too deep and causing the death of the rat, thereby effectively improving the success rate of modeling; and avoiding accidental injury of the pons or medulla oblongata by the needle, which affects the accuracy of cognitive function observation.

[0024] 2. In the present invention, the position where the ultrasonic probe shows the clearest cistern of the rat is marked on the occipital neck before modeling, and the best puncture area is determined so that the puncture position can be quickly found during modeling.

[0025] 3. In the process of modeling, a limiting sleeve is provided on the puncture needle to ensure that only the 4 mm long needle tip can puncture the skin, which can prevent the needle from entering too shallowly or too deeply, and effectively improve the success rate of the model.

[0026] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of puncturing the cistern magna under ultrasound guidance in an embodiment of the present invention;

[0028] Figure 2 The brain images of rats in the experimental group and the sham operation group in the embodiment of the present invention, wherein A is the experimental group and B is the sham operation group;

[0029] Figure 3 This is the T2flair image of the rats in the experimental group in the embodiment of the present invention;

[0030] Figure 4 ADC images of rats in the experimental group in the embodiment of the present invention;

[0031] Figure 5 1 is a diagram of brain nerve fibers of rats in the experimental group in the embodiment of the present invention. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0033] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following examples are usually measured in accordance with national standards. The experimental instruments, equipment and reagents without source indication in the following examples are all commercially available raw materials.

[0034] The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the common international standards, conventional conditions, or conditions recommended by the manufacturer.

[0035] Unless otherwise specified, all parts are by weight and all percentages are by mass percentage. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equivalent to the described content can be applied to the method of the present invention. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0036] The experimental animals in the following examples were SD rats or Wistar rats, and the breeding and treatment of rats complied with the animal welfare and ethical guidelines of the Anhui Provincial Laboratory Animal Management Committee.

[0037] The statistical methods in the following examples were analyzed using GraphPad Prism 5 software. The survival rate and mortality rate of each group of suckling mice were evaluated using Mantel-Cox log rank software.

[0038] Example

[0039] Application of rat SAH model in cognitive function research, the construction method of rat SAH model is as follows:

[0040] Step 1, selecting normal rats, marking the modeling position on the occipital and cervical regions of the rats, and performing SAH modeling on the rats under ultrasound guidance to obtain a rat SAH model;

[0041] Step 2, performing ultrasound scanning on the rat SAH model constructed in step 1 and the sham operation group to verify whether the rat SAH model is successfully established;

[0042] Step 3, performing magnetic resonance imaging on the rat SAH model constructed in step 1 and the sham operation group to verify whether the cognitive function of the rat SAH model is affected;

[0043] Step 4, observing the brain nerve fibers of the rats in the SAH model and sham operation group constructed in step 1.

[0044] The detailed process of step 1 is as follows ( Figure 1 To puncture the cisterna magna under ultrasound guidance):

[0045] The rat was placed in a prone position on a foam pad, with the head naturally hanging down over the edge of the pad and resting on the table to form a 45°-60° tilt angle. First, the ultrasound probe was placed at the cistern magna, keeping the needle at an upward angle of 60-80° to find the cistern, and then a 1 mm inner diameter limiting sleeve was placed on the puncture needle to ensure that only a 4 mm length of the needle tip could penetrate the skin. The 4 mm length can prevent the needle from entering too shallowly or too deeply, effectively improving the success rate of the model.

[0046] Fix the skin of the rat's head and neck, use a 26G puncture needle to puncture from the marked puncture point at an angle parallel to the head and aligned with the line from the external auditory canal, and observe whether there is a high echo position in the occipital cistern by ultrasound, then advance the needle in a small amplitude, and inject a small dose of blood to observe whether there is blood in the occipital cistern. If blood is injected, it indicates that the position is correct and the rat SAH model is successfully established. Ultrasound scanning is used for exploration, and a high-frequency ultrasound probe is used during exploration, and the frequency of the ultrasound probe is 10-12MHz. Figure 2 These are the brain images of rats in the experimental group and the sham operation group.

[0047] Magnetic resonance imaging was performed on the constructed rat SAH model and the sham operation group to verify whether the cognitive function of the rat SAH model was affected. Scanning was performed using a soft coil and a fast echo DTI sequence scan with the following scanning parameters: TR = 2605ms, TE = 30.85ms, four segments, b = 1000s / mm 2 , 30diffusion directions, five B0images, matrix=128×128pixels2, FOV=30×30mm 2 , slice thickness = 2.0 mm;

[0048] T2Flair sequence: TE = 6000ms, TR = 102ms, Matrix = 256 × 256 × 128 pixels3,

[0049] FOV = 20 × 20 × 20 mm 3 ; slice thickness = 2.0 mm;

[0050] DWI sequence: TE = 3990 ms, TR = 56 ms, Matrix = 256 × 256 × 128 pixels3, FOV = 20 × 10 × 20 mm 3 ;slice thickness=2.0mm.

[0051] T2flair images of rats in the experimental group are shown in Figure 2. Figure 3 As shown, the ADC image is as follows Figure 4 shown.

[0052] 48 hours after modeling, magnetic resonance DTI imaging was performed and FA values ​​were calculated, as shown in Table 1:

[0053] Table 1

[0054]

[0055] As shown in Table 1, the FA values ​​of the frontal lobe, temporal lobe and hippocampus of the rats in the experimental group were significantly reduced, indicating that the white matter microstructure of the rats was damaged, further indicating that the rat SAH model was successfully established and the model rats had cognitive dysfunction.

[0056] After 12 hours of modeling, magnetic resonance diffusion imaging was performed and the ADC values ​​were calculated as shown in Table 2 (unit: ×10 -3 mm 2 / s):

[0057] Table 2

[0058]

[0059] As shown in Table 2, before and after modeling in the experimental group, the ADC values ​​of the bilateral frontal cortex, bilateral temporal cortex and bilateral hippocampus of the rats were significantly reduced, indicating that the white matter microstructure of the rats was damaged, further indicating that the rat SAH model was successfully constructed and the model rats had cognitive dysfunction.

[0060] The rat SAH model was constructed to observe the rat brain nerve fibers. Figure 5 shown.

[0061] Therefore, the present invention adopts the above-mentioned rat SAH model in the study of cognitive function, and the constructed rat SAH model has good stability, high success rate, obvious model behavior, and can be used for the study of diseases such as cognitive impairment.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Application of the rat SAH model in cognitive function research, characterized in that: The method for establishing the rat SAH model is as follows: Step 1, selecting normal rats, marking the modeling position on the occipital and cervical regions of the rats, and performing SAH modeling on the rats under ultrasound guidance to obtain a rat SAH model; Step 2, performing ultrasound scanning on the rat SAH model constructed in step 1 and the sham operation group to verify whether the rat SAH model is successfully established; Step 3, performing magnetic resonance imaging on the rat SAH model constructed in step 1 and the sham operation group to verify whether the cognitive function of the rat SAH model is affected; Step 4, observing the brain nerve fibers of the rats in the SAH model and sham operation group constructed in step 1.

2. The use of the rat SAH model according to claim 1 in cognitive function research, characterized in that: When performing the rat SAH modeling under ultrasound guidance in step 1, a high-frequency ultrasound probe is used, and the frequency of the ultrasound probe is 10-12 MHz.

3. The use of the rat SAH model according to claim 1 in cognitive function research, characterized in that: In step 3, the magnetic resonance scan was completed using a fast echo DTI sequence scan with the following scanning parameters: TR = 2605 ms, TE = 30.85 ms, four segments, b = 1000 s / mm 2 , 30diffusion directions, five B0 images, matrix=128×128pixels2, FOV=30×30mm 2 , slice thickness = 2.0 mm; T2Flair sequence: TE = 6000ms, TR = 102ms, Matrix = 256 × 256 × 128 pixels3, FOV=20×20×20mm 3 ;slice thickness=2.0mm; DWI sequence: TE = 3990 ms, TR = 56 ms, Matrix = 256×256×128 pixels3, FOV = 20×10×20 mm 3 ; slice thickness = 2.0 mm.

4. The use of the rat SAH model according to claim 1 in cognitive function research, characterized in that: Applied to cognitive function research.

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