CN118490406A - Novel method for constructing animal model of acute ischemic cerebrovascular disease - Google Patents
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Abstract
The invention relates to the technical field of cerebral small vessel diseases, in particular to a novel method for constructing an animal model of acute ischemic cerebral small vessel diseases. The traditional method for constructing the animal model of the acute ischemic cerebrovascular disease has the problems of poor accuracy, poor repeatability and the like. In order to solve the problems, the invention utilizes the attention mechanism to optimize the key parameters for constructing the animal model, and the optimization process comprehensively considers objective indexes such as infarct volume, neurological deficit score, blood brain barrier permeability and the like. The validity of the model is judged by integrating the evaluation modes of magnetic resonance imaging, behavioral testing, histopathological examination and the like. Compared with the traditional method, the method can obviously improve the accuracy and the repeatability of the construction of the animal model of the acute ischemic cerebrovascular disease.
Description
Technical Field
The invention relates to the technical field of cerebral small vessel diseases, in particular to a novel method for constructing an animal model of acute ischemic cerebral small vessel diseases.
Background
Cerebral arteriole disease (Cerebral SMALL VESSEL DISEASE, CSVD) is a common age-related cerebrovascular disease that affects primarily small blood vessels such as arterioles, capillaries and venules in the brain, resulting in a range of clinical, imaging and pathological changes. CSVD can be divided into two types of Acute morbidity and chronic progress, wherein Acute ischemic cerebral small vascular diseases (Acute ISCHEMIC CSVD, AI-CSVD) take Acute cerebral infarction as main clinical manifestation, often accompanied by sequelae such as neurological impairment and cognitive dysfunction, and seriously affecting the life quality of patients.
Although the pathogenesis of AI-CSVD has not been fully elucidated, studies have shown that structural and functional abnormalities of cerebral small blood vessels may be the major pathological basis. To further investigate the pathogenesis of AI-CSVD and to find potential therapeutic strategies, it is necessary to construct a suitable animal model.
Currently, the construction method of CSVD animal models mainly comprises a spontaneous hypertension rat model, a bilateral common carotid artery stenosis model, a genetically modified animal model and the like. The models simulate the pathological characteristics of CSVD to a certain extent, but have some defects such as long model construction period, high operation difficulty, lack of evaluation indexes and the like. Therefore, development of a new animal model construction method is needed to improve the accuracy and reliability of the model.
In recent years, artificial intelligence technology has been increasingly used in medical research, where attention mechanisms are receiving a great deal of attention due to their strong feature extraction and sequence modeling capabilities. Considering that the AI-CSVD animal model construction process involves various parameters and evaluation indexes, the method cooperates with the computer college of the school to introduce an attention mechanism into the field, and on the basis, the BrainFormer for constructing the animal model of the ischemic cerebrovascular disease is provided, and particularly the efficiency and the accuracy of model construction are improved by integrating objective indexes such as infarct volume, neurological deficit, blood brain barrier permeability and the like.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a novel method for constructing an animal model of acute ischemic cerebrovascular disease, which comprises the following steps:
step 1: animal preparation, 10 healthy adult male C57BL/6J mice (8-10 weeks old) were selected;
Step 2: anesthesia and body temperature monitoring: mice were given either allo-inhalation or injection anesthesia according to BrainFormer optimized anesthesia mode parameters. The body temperature of the mice was monitored using a rectal thermometer and maintained at 37.0-37.5 ℃ with a heated plate.
Step 3: head fixation and skin disinfection: the anesthetized mice were mounted on a stereotactic apparatus, head hair was shaved, and scalp was sterilized with iodophor solution.
Step 4: craniotomy and dura mater exposure: about 1.5cm was cut centrally along the scalp, revealing the skull. A dental drill was used to drill a bone window of approximately 2mm diameter into the skull, exposing the dura mater.
Step 5: stereotactic and drug injection: according to BrainFormer optimized injection position parameters, adjusting a stereotactic instrument, slowly inserting a microinjector into a target brain region, and according to optimized injection medicine concentration, volume and rate parameters, injecting an L-NIO solution into brain tissue to cause focal ischemic injury;
step 6: wound suturing and anesthesia recovery: after the injection is finished, slowly withdrawing the injector, sealing the skull defect with medical glue, and suturing the scalp. The mice are moved to a constant temperature heating plate to wait for autonomous awakening;
Step 7: magnetic resonance imaging examination: the mice are subjected to magnetic resonance imaging examination, which comprises sequences of T2 weighted images, diffusion weighted images and the like, and the structural and functional changes of brain tissues are estimated;
Step 8: monitoring and evaluation: performing neurological deficit scoring and open field experiments on the mice to evaluate changes in motor, sensory, cognitive and other functions;
Step 9: animal sacrifice and specimen collection: mice were sacrificed and brains were taken for histopathological examination, including HE staining, nissl staining, and GFAP immunofluorescence staining, to assess morphological changes in brain tissue.
The invention introduces an attention mechanism into the field of AI-CSVD animal model construction and constructs BrainFormer for creating an acute ischemic cerebrovascular disease animal model based on the attention mechanism, and compared with the traditional method, the method has the following two main advantages:
(1) Optimization of key parameters is achieved: modeling and learning are carried out on drug injection parameters, operation parameters and the like through BrainFormer, and particularly, objective indexes such as the volume of the integrated infarction, the neurological deficit, the blood brain barrier permeability and the like are comprehensively utilized to obtain an optimal parameter combination, so that blindness and subjectivity are avoided, the accuracy and the repeatability of model construction are improved, and animal models which integrate the objective indexes are rarely available in the existing method;
(2) An objective model evaluation system is established: by integrating a plurality of indexes such as magnetic resonance imaging, nerve function scoring, histopathological examination and the like, objective judgment of the effectiveness of the animal model is realized.
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FIG. 1 is a block flow diagram of the steps of the present invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
As shown in FIG. 1, the novel method for constructing the animal model of the acute ischemic cerebrovascular disease provided by the invention comprises the following steps:
step 1: preparing animals; step 2: anesthesia and body temperature monitoring; step 3: head fixation and skin disinfection; step 4: craniotomy and dura mater exposure; step 5: stereotactic positioning and drug injection; step 6: wound suturing and anesthesia recovery; step 7: magnetic resonance imaging examination; step 8: monitoring and evaluating; step 9: animal sacrifice and specimen collection.
Specifically, the invention selects healthy adult male C57BL/6J mice (8-10 weeks old) as experimental animals.
Mice were randomly divided into the following four groups:
(1) Sham group (Sham group): only craniotomy is performed, no drug injection is given, n=10;
(2) Model group (Model group): constructing an AI-CSVD model according to a traditional method, namely, injecting L-NIO in a stereotactic way to induce acute cerebral ischemia, wherein n=10;
(3) BrainFormer parameter optimization group (BrainFormer-P group): constructing an AI-CSVD model by adopting parameters optimized based on BrainFormer, wherein n=10;
(4) BrainFormer evaluate optimization group (BrainFormer-E group): an AI-CSVD model was constructed using parameters optimized based on BrainFormer, n=10.
To optimize key parameters in the construction of the AI-CSVD animal model, brainFormer was introduced in this study. In this study, we consider the parameters in the AI-CSVD model building process as a sequence, which was modeled and optimized by BrainFormer.
First, we define the following parameters:
(1) Concentration of injected drug (Injection Drug Concentration, IDC): refers to the concentration of the stereotactic injection L-NIO solution, the unit is mg/mL, recorded as ;
(2) Injection Volume (IV): refers to the volume of the stereotactic injection L-NIO solution, expressed in μL, recorded as;
(3) Injection Rate (IR): the rate of stereotactic injection of L-NIO solution in μL/min is indicated and is recorded as;
(4) Injection Site (IS): the brain position of the stereotactic injection L-NIO solution is indicated by brain stereotactic map coordinates and is recorded as;
(5) Anesthesia mode (ANESTHESIA METHOD, AM): refers to an anesthesia method adopted in the animal model construction process, which comprises two steps of inhalation anesthesia (Inhalation Anesthesia, IA) and injection anesthesia (Injection Anesthesia, injA), respectively recorded asAnd;
(6) Body temperature control (Body Temperature Control, BTC): refers to whether the temperature control measure is adopted in the animal model construction process, and is recorded asA value of 0 or 1,0 indicating no adoption, and 1 indicating adoption.
Thus, we can represent the construction process of each animal model as a sequence of parameters:
;
、、、、、、 as a continuous variable, the number of the variables, 、Is a binary variable.
In the present invention, we use self-attention to model parameter sequencesThe interaction between the parameters of the system.
Specifically, for parameter sequencesWe first convert it into an embedded vector sequence:
;
For the length of the sequence,Is the embedding dimension.
We then calculate self-attention by:
;
、、 each of which is a query, key, value matrix, respectively, The dimensions for the key matrix can be obtained by linear transformation:
;
wherein the method comprises the steps of ,,Is a weight matrix that can be learned.
By self-attention we can get a sequence of parametersAttention weights of each parameter in (c) to capture its impact on model building effects.
To further increase the expressive power of the model, we further employ multi-head attention, i.e. input embedded vectors to the model respectivelyFrom different self-attention modules, we getAnd (3) outputting and splicing the two components:
;
;
、、、 is a weight matrix that can be learned.
Through the multi-head attention, the model can learn multiple interaction modes among parameters in different subspaces, and the effect of parameter optimization is improved.
After self-attention, the features are non-linearly transformed:
;
、、、 Is a learnable weight matrix and a bias vector.
By introducing nonlinear transformation, the model can learn more complex interaction modes among parameters, and the optimization effect is improved.
To facilitate convergence and generalization, brainFormer employs LayerNorm and residual connections after each sub-layer:
;
;
、 The mean and standard deviation of the features respectively, 、Is a learnable scaling and offset parameter.
For BrainTransformer, we designed the following loss function:
;
for infarct volume loss:
;
is the first The true infarct volume of the individual samples,Infarct volume obtained for the model.
For neurological deficit loss, using mNSS scoring calculations:
;
is the first The true mNSS score of each sample,MNSS scores were obtained for the model.
For blood brain barrier permeability loss, evans Blue staining was used to calculate:
;
is the first The true Evans Blue leakage of the individual samples,Evans Blue leakage was obtained for the model.
Is a regularization term:
;
The number of layers for the model. 、、Is a balancing factor for controlling the weight of each loss term.
In the model training process, an Adam optimizer is adopted to update model parameters:
;
As a parameter of the model, it is possible to provide, Is the learning rate.
By design of a loss function and model training, an optimal parameter combination can be obtained for subsequent AI-CSVD model construction.
After obtaining the optimal parameter combinations, we constructed an AI-CSVD animal model according to the following steps:
Anesthesia and operation preparation, and the mice are anesthetized according to the anesthesia mode parameters obtained through BrainFormer optimization. If inhalation anesthesia is adopted ) Inhalation induction and maintenance was performed using isoflurane (1.5-2.0%); if injection anesthesia is adopted) The mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) was injected intraperitoneally.
The anesthetized mice were fixed on a stereotactic apparatus (RWD, china) and the eye ointment was applied to both eyes to prevent corneal dryness. If in the optimized parametersThe body temperature of the mice was monitored using a rectal thermometer and maintained at 37.0-37.5 ℃ by a murine thermostatted blanket (RWD, china).
Stereotactic and skull drilling: optimized injection position parameters according to BrainFormerThe stereotactic instrument was adjusted to determine the L-NIO injection site. Approximately 1.5cm was cut centrally in the scalp of the mice, exposing the skull. A bone window of about 2mm in diameter was drilled into the surface of the skull using an electric sander, exposing the dura mater.
The microinjection pump is then connected to the microinjector with a loading concentration ofMg/mL of L-NIO solution. The resulting injection volume is optimized according to BrainFormerAnd injection rateSlowly pushing the injector under the skullMm, in order toSpeed injection in mu L/minMu L L-NIO solution, after the injection is finished, the needle is left for 5min, and the syringe is slowly pulled out.
The skull defect is sealed by using medical glue, the scalp is sutured, and iodophor is smeared for disinfection. The mice were moved to a thermostatically heated plate for resuscitation until they were able to move autonomously. The sham operated mice were subjected to only craniotomy, and an equal volume of physiological saline was injected into the brain.
To evaluate brain damage in the AI-CSVD model, we examined the mice on magnetic resonance imaging 24h after drug injection. Prior to the scan, mice were anesthetized by inhalation of 2% isoflurane and then maintained under 1-1.5% isoflurane mixed oxygen inhalation to maintain anesthesia.
Considering the impact of scan parameters on image quality and brain injury assessment, we use the following scan parameters:
;
TR is repetition time, TE is echo time, FA is flip angle, NS is scan number of layers, VS is voxel size.
After the scan was completed, we used ITK-SNAP software to manually delineate and measure volume of infarct foci on T2 weighted images. The diffusion weighted image is preprocessed using SPM12 software package, including head movement correction, eddy current correction, brain extraction, etc. We then calculated the anisotropy scores (Fractional Anisotropy, FA) and the average diffusion coefficients (Mean Diffusivity, MD) using the FDT tool in FMRIB software library.
To quantitatively evaluate the brain injury level of each group of mice, we measured the following index:
;
Next, we performed a neurological deficit score (Modified Neurological Severity Score, mNSS) to evaluate the neurological function of each group of mice. The mNSS score includes four aspects of motor, sensory, reflex and balance, with a total score of 18 points, with higher scores indicating more severe neurological deficit.
We scored mNSS on day before surgery, day 1, day 3, day 7 after surgery, and averaged three consecutive scores. The results were as follows:
;
To assess spontaneous locomotor ability and anxiety levels in mice, we performed open field experiments on each group of mice on day 7 post-surgery. The experiment was carried out in a 50cm by 40cm black open plastic box, the bottom of which was evenly divided into 25 10cm by 10cm squares. The mice were placed in the center of the open field, their movement tracks were recorded using a video camera, and each mouse was tested for 10min.
The following index was measured:
;
At the end of the behavioural experiment, we euthanized each group of mice, decapitated the brain, and brain tissue was immersed in 4% paraformaldehyde for fixation for 24h. The fixed brain tissue was dehydrated with 30% sucrose solution, embedded in OCT frozen embedding medium, and the brain tissue was coronally cut into serial sections of 20 μm thickness using a frozen microtome.
We stained brain sections using the following staining method:
(1) Hematoxylin-eosin staining: conventional paraffin section, dewaxing to water, hematoxylin staining for 5min, hydrochloric acid alcohol differentiation, eosin staining for 2min, gradient alcohol dehydration, xylene transparency, and neutral resin sealing. HE staining may reveal the basic morphological structure of brain tissue.
(2) Nissl staining: frozen sections, 0.1%Cresyl violet staining for 10min, PBS washing, gradient alcohol dehydration, xylene transparency, neutral resin sealing. Nissl staining can specifically reveal the cell bodies and nucleoli of neurons.
(3) Immunofluorescent staining: frozen sections, PBS washes, 5% goat serum blocked for 1h at room temperature, primary antibody incubated overnight at 4 ℃, secondary antibody incubated for 1h at room temperature, DAPI counterstain for 5min, anti-fluorescence quenching blocked tablet blocking. The primary antibody is rabbit anti-GFAP and marks astrocytes; the secondary antibody was Alexa Fluor 488 conjugated goat anti-rabbit IgG.
The stained sections were photographed using a positive fluorescence microscope, 5 fields were randomly selected per section, and 3-5 images at different focal planes were acquired per field. Processing and analyzing the image, and measuring to obtain the following indexes:
;
through BrainFormer parameter optimization, we obtained a set of optimal parameter combinations for subsequent AI-CSVD animal model construction:
Construction of optimal parameters for animal models
;
To evaluate the effect of parameter optimization, we constructed AI-CSVD animal models using pre-optimization (i.e., traditional method) and post-optimization parameters, respectively, and compared infarct volume, mNSS score, and Evans Blue leakage for both models. The result shows that the optimized parameter combination can obviously reduce the infarct volume, improve the neurological deficit, reduce the blood brain barrier permeability, and prompt BrainFormer can effectively optimize the construction process of an AI-CSVD animal model and improve the accuracy and the repeatability of the model.
Infarct volume comparison of AI-CSVD animal models before and after parameter optimization:
;
comparison of mNSS scores of AI-CSVD animal models before and after parameter optimization:
;
Evans Blue leakage amount comparison of AI-CSVD animal models before and after parameter optimization:
;
By means of magnetic resonance T2 weighted imaging, we found that model group mice showed significantly higher signal lesions at the site of drug infusion, suggesting the presence of acute ischemic injury, compared to sham-operated groups. Further quantitative analysis shows that the infarct volume of mice in BrainFormer parameter optimization group and evaluation optimization group is significantly smaller than that of mice in model group but larger than that of mice in sham operation group, which suggests that the brain injury degree of AI-CSVD mice can be significantly reduced by the BrainFormer optimization-based method.
Qualitative analysis results of the magnetic resonance T2 weighted images of mice of each group:
;
infarct volume quantitative analysis results of the magnetic resonance T2 weighted images of mice of each group:
;
Diffusion weighted imaging results show that compared with a sham operation group, the ischemia area of the mice in the model group shows obvious diffusion limitation, the FA value is obviously reduced, the MD value is obviously increased, and the existence of angiogenic edema and apoptosis necrosis is indicated. And the FA value of mice in BrainFormer parameter optimization groups and evaluation optimization groups is higher than that of mice in model groups, the MD value is lower than that of mice in model groups, and the damage of brain tissue microstructure and the degree of edema are indicated to be lighter.
FA value quantitative analysis results of the magnetic resonance diffusion weighted images of the mice in each group:
;
quantitative analysis results of MD values of the magnetic resonance diffusion weighted images of the mice in each group:
;
The results of the mNSS scores showed that the scores of the sham mice at each time point were close to 0, suggesting that their neurological functions were normal. The other three groups of mice all showed different degrees of neurological deficit after surgery, as evidenced by significantly higher mNSS scores than the sham-operated group. The neurological deficit was most severe in the model mice, and the post-operative day 1 mNSS score reached (12.5.+ -. 1.8) and recovery was slow, with (9.3.+ -. 1.5) still at day 7. The BrainFormer parameter optimized group and the evaluation optimized group mice were relatively light in neurological deficit, and the post-operative day 1 mNSS scores were (8.2.+ -. 1.2) and (7.8.+ -. 1.3) respectively, and recovered to (4.5.+ -. 1.0) and (4.1.+ -. 0.9) respectively on day 7, which were significantly different from the model group, but still higher than the sham-operated group.
Results of mNSS scoring for each group of mice:
;
The results of the open field experiments show that compared with the sham operation group, the total moving distance of the mice in the model group is obviously reduced, the residence time in the central area is obviously shortened, the number of erection times is obviously reduced, the spontaneous movement capacity is reduced, the anxiety level is increased, and the exploratory behavior is reduced. However, each index of BrainFormer parameter optimization group and evaluation optimization group mice is superior to the model group, but still has differences with the sham operation group.
Open field experimental results for each group of mice:
;
HE staining results showed that the brain tissue structure of sham mice was intact and no obvious pathological changes were seen. The injection sites of the other three groups of mice can see ischemic necrosis sites with different degrees, and the ischemic necrosis sites are expressed as nucleus shrinkage, cytoacidophilic denaturation and tissue loosening. Quantitative analysis shows that the infarct area percentage of the mice in the model group reaches (28.5+/-4.2)%, which is obviously higher than that of the mice in the sham operation group. The percent infarct sizes of BrainFormer parameter-optimized and evaluation-optimized groups were (15.2±2.6)% and (13.9±2.3)%, respectively, significantly lower than the model group, but still higher than the sham-operated group.
Qualitative analysis results of HE staining for each group of mice:
;
results of quantitative analysis of percentage infarct size for HE staining in each group of mice:
;
Nissl staining results showed that the ischemia zone survival neuron density of sham mice was (1325.+ -.118) per mm . While the surviving neuron density of the remaining three groups of mice was significantly lower than that of the sham-operated group, with the model group being lowest (415.+ -.84) pieces/mmBrainFormer parameter optimization groups and evaluation optimization groups are (782+/-102) pieces/mm respectivelyAnd (825.+ -.95) pieces/mmSignificantly higher than the model set.
Quantitative analysis of the Nissl-stained viable neuron density in each group of mice:
;
The result of GFAP immunofluorescence staining shows that the density of GFAP positive astrocytes in the ischemia zone of the sham mice is (65+ -12) pieces/mm . Whereas the GFAP positive cell density was significantly higher in the remaining three groups of mice than in the sham group, with model groups up to (352±58) pieces/mmBrainFormer parameter optimization group and evaluation optimization group were (218.+ -.43) pieces/mm, respectivelyAnd (195.+ -.39) pieces/mmSignificantly lower than the model set.
Quantitative analysis of positive astrocyte density by GFAP immunofluorescent staining for each group of mice:
。
Claims (7)
1. The method for constructing the novel acute ischemic cerebrovascular disease animal model is characterized by comprising the following steps of:
step 1: animal preparation, 10 healthy adult male C57BL/6J mice (8-10 weeks old) were selected;
Step 2: anesthesia and body temperature monitoring: according to BrainFormer optimized anesthesia mode parameters, the mice are subjected to different inhalation anesthesia or injection anesthesia, and a rectal thermometer is used for monitoring the body temperature of the mice;
Step 3: head fixation and skin disinfection: fixing the anesthetized mice on a stereotactic instrument, shaving head hair, and sterilizing scalp with iodophor solution;
Step 4: craniotomy and dura mater exposure: incision about 1.5cm along the centre of the scalp, revealing the skull; drilling a bone window with the diameter of about 2mm on the skull by using a dental drill, and exposing the dura mater;
Step 5: stereotactic and drug injection: according to BrainFormer optimized injection position parameters, adjusting a stereotactic instrument, slowly inserting a microinjector into a target brain region, and according to optimized injection medicine concentration, volume and rate parameters, injecting an L-NIO solution into brain tissue to cause focal ischemic injury;
Step 6: wound suturing and anesthesia recovery: after the injection is finished, slowly withdrawing the injector, sealing the skull defect with medical glue, suturing the scalp, moving the mouse to a constant temperature heating plate, and waiting for the mouse to wake up independently;
step 7: magnetic resonance imaging examination: performing magnetic resonance imaging examination on the mice;
step 8: monitoring and evaluation: performing neurological deficit scoring and open field experiments on the mice to evaluate changes in motor, sensory, and cognitive functions;
step 9: animal sacrifice and specimen collection: mice were sacrificed and brains were taken for histopathological examination.
2. The method of claim 1, wherein the BrainFormer-optimized parameters areThe method specifically comprises the following steps:
(1) Concentration of injected drug: refers to the concentration of the stereotactic injection L-NIO solution, the unit is mg/mL, recorded as ;
(2) Injection volume: refers to the volume of the stereotactic injection L-NIO solution, expressed in μL, recorded as;
(3) Injection rate: the rate of stereotactic injection of L-NIO solution in μL/min is indicated and is recorded as;
(4) Injection position: the brain position of the stereotactic injection L-NIO solution is indicated by brain stereotactic map coordinates and is recorded as;
(5) Anesthesia mode: the method for anesthesia adopted in the animal model construction process comprises inhalation anesthesia and injection anesthesia, which are respectively marked asAnd;
(6) Body temperature control: refers to whether the temperature control measure is adopted in the animal model construction process, and is recorded asA value of 0 or 1,0 indicating no adoption, and 1 indicating adoption.
3. The method of claim 2, wherein the specific parameters are as follows:
Concentration of injected L-NIO solution: 30mg/mL; volume of injected L-NIO solution: 0.6. Mu.L; rate of injection of L-NIO solution: 0.1. Mu.L/min; injection position: (2.0 mm, 0.5mm, 3.0 mm); anesthesia mode: isoflurane inhalation anesthesia; body temperature control: is the result.
4. The method of claim 3, wherein said monitoring and assessing, for neurological deficit scoring and open field testing, the motor, sensory, cognitive function alterations in mice comprises: experiments were carried out in a 50cm x 40cm black open plastic box, the bottom of the box was evenly divided into 25 10cm x 10cm squares, the mice were placed in the center of the open field, their activity tracks were recorded using a video camera, and each mouse was tested for 10min.
5. A method according to claim 3, characterized in that the magnetic resonance imaging examination specifically comprises: the T2 weighted image, the T1 weighted image and the diffusion weighted image sequence are used for evaluating the structural and functional changes of the brain tissue.
6. The method of claim 3, wherein the animal is sacrificed and specimens collected: mice were sacrificed and brains were taken for histopathological examination specifically including: each group of mice was euthanized, brain tissue was immersed in 4% paraformaldehyde for 24h fixation, the fixed brain tissue was dehydrated with 30% sucrose solution, embedded in OCT frozen embedding medium, and the brain tissue was coronally cut into serial sections 20 μm thick using a frozen microtome, and the brain sections were stained using the following staining method:
(1) Hematoxylin-eosin staining: conventional paraffin section, dewaxing to water, hematoxylin staining for 5min, hydrochloric acid alcohol differentiation, eosin staining for 2min, gradient alcohol dehydration, xylene transparency, neutral resin sealing;
(2) Nissl staining: freezing, slicing, 0.1%Cresyl violet staining for 10min, washing with PBS, gradient alcohol dehydrating, transparent xylene, and sealing with neutral resin;
(3) Immunofluorescent staining: freezing, washing with PBS, sealing 5% goat serum at room temperature for 1h, incubating the primary antibody at 4 ℃ overnight, incubating the secondary antibody at room temperature for 1h, counterstaining with DAPI for 5min, sealing the anti-fluorescence quenching sealing tablet, wherein the primary antibody is rabbit anti-GFAP, and labeling astrocytes; the secondary antibody was Alexa Fluor 488 conjugated goat anti-rabbit IgG.
7. The method of claim 1, wherein the BrainFormer penalty is:;
for infarct volume loss:
;
is the first The true infarct volume of the individual samples,Infarct volume obtained for the model;
For neurological deficit loss, using mNSS scoring calculations:
;
is the first The true mNSS score of each sample,MNSS score obtained for the model;
For blood brain barrier permeability loss, evans Blue staining was used to calculate:
;
is the first The true Evans Blue leakage of the individual samples,Evans Blue leakage obtained for the model;
Is a regularization term:
;
The number of layers to be the model is, 、、Is a balancing factor for controlling the weight of each loss term.
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