阅读说明：本技术 一种小鼠高血压脑出血模型的造模方法 (Modeling method of mouse hypertensive cerebral hemorrhage model ) 是由 胡泊 荣良群 张清秀 于 2021-08-18 设计创作，主要内容包括：本发明公开了一种小鼠高血压脑出血模型的造模方法,所述方法步骤如下：首先在小鼠的左心室穿刺取血,然后通过擦拭清理覆盖颅骨的软组织暴露颅骨术野,确定颅骨表面进针点后进行钻孔,再通过脑立体定位仪于钻孔处注射自体血,完成注射后,用骨蜡封住钻孔,消毒切口,缝合皮肤,即可。本发明采取左心室穿刺取动脉血,能在提取足量动脉血的前提下不降低小鼠存活率,通过小鼠脑定位仪精准地将自体动脉血回注入脑内基底节区尾壳核,形成稳定的血肿,为后续的实验提供保障,利于实验对照研究,且与临床上高血压患者病理变化和疾病转归极为相似,对脑出血后的疾病转归及治疗方案具有重要意义。(The invention discloses a molding method of a mouse hypertensive cerebral hemorrhage model, which comprises the following steps: firstly, puncturing the left ventricle of a mouse to take blood, then cleaning soft tissues covering the skull by wiping to expose a skull surgical field, drilling holes after determining a needle inserting point on the surface of the skull, then injecting autoblood at the drilling holes by a brain stereotaxic apparatus, sealing the drilling holes by bone wax after completing injection, disinfecting incisions, and suturing the skin. According to the invention, the arterial blood is obtained by puncturing the left ventricle, the survival rate of the mouse can not be reduced on the premise of extracting sufficient arterial blood, the autologous arterial blood is accurately injected back into the caudal putamen of the basal ganglia region in the brain through the mouse brain positioning instrument to form stable hematoma, the follow-up experiment is guaranteed, the experimental contrast research is facilitated, the pathological change and the disease outcome of the clinical hypertension patient are very similar, and the method has important significance for the disease outcome and the treatment scheme after cerebral hemorrhage.)

1. A molding method of a mouse hypertensive cerebral hemorrhage model is characterized by comprising the following steps:

step 1) left ventricle puncture blood sampling: the mouse is anesthetized by isoflurane gas induction, the mouse is placed in a supine position, and four limbs of the mouse are fixed, so that the chest and the upper abdomen of the mouse are fully exposed; sterilizing the chest and the upper abdomen of the mouse in a large range, and then puncturing and drawing blood at the included angle between the xiphoid process of the sternum and the costal bone by inclining 10-20 degrees to the apex of the heart to rapidly transfer arterial blood for later use;

step 2) exposure of the cranioplasty field: making a 1cm long midline incision on the scalp of a mouse with a scalpel, wiping and cleaning the soft tissue covering the skull to expose the vertical intersection point, i.e. the coronal and sagittal suture intersection point;

step 3), determining a skull surface needle insertion point: mounting the microsyringe on an injection pump, and stereoscopically orienting a needle head to a coronal and sagittal suture intersection point and zeroing; next, the stereotactic manipulator arm was adjusted to place the needle 0.2mm in front of the intersection point of the coronal and sagittal sutures and 2mm lateral to the right; at this coordinate a variable speed drill was used to drill a hole in the skull of the mouse;

step 4), injecting autoblood by using a brain stereotaxic apparatus: rapidly extracting the arterial blood reserved in the step 1), transferring the arterial blood into a glass cylinder of a microsyringe, pushing a needle head to the ventral side by 3.7mm through a drill hole on the surface of the skull of the mouse, and injecting autologous arterial blood to the caudal putamen of the right side;

step 5) standing the microsyringe: after injection is finished, the needle head is left in the original position for 10min, then the needle head is drawn back at the speed of 1mm/min, blood returning of the puncture channel is reduced, and the drilled hole is sealed by bone wax;

and 6) disinfecting the incision and suturing the skin.

2. The method for modeling a mouse model of hypertensive cerebral hemorrhage according to claim 1, wherein the mouse is a KM mouse and is derived from swiss mouse.

3. The modeling method of mouse model of hypertensive cerebral hemorrhage according to claim 1, wherein the injection rate of the autologous arterial blood of step 4) is 2 μ L/min.

4. The method as claimed in claim 1, wherein when suturing the skin in step 6), the suture site is properly injected with saline solution to make a replacement for the mouse's abdominal cavity.

5. The modeling method of a mouse model of hypertensive cerebral hemorrhage according to claim 1, further comprising step 7): and (3) evaluating the mice by adopting an additive removal test or scoring the neurological deficit 24h after operation, wherein the mice meeting the evaluation/scoring standard are the mice successfully modeled.

6. The modeling method of mouse model of hypertensive cerebral hemorrhage according to claim 5, wherein the step of the additive removal test is as follows: attaching 0.3cm × 0.4cm of adhesive tape to the injured forepaw on the opposite side of the mouse; measuring the tactile motion response by recording the time for removing the adhesive tape, wherein the maximum observation time is 120 s; mice successfully molded are obtained when the time for removing the adhesive tape is more than 10 s.

7. The modeling method of mouse model of hypertensive cerebral hemorrhage according to claim 5, wherein the scoring of neurological deficit comprises the following steps:

(1) placing the mouse on the surface of an empty platform to freely move for 2-5 min;

(2) placing the mouse on a 45-degree grid inclined plane for 60 s;

(3) lifting the tail of the mouse and standing upside down for 30 s;

(4) lifting the tail of the mouse and standing upside down, and placing the forelimb of the mouse on a table top for 30 s;

the scoring criteria for the neurological deficit score are as follows:

for step (1): the body symmetry is normal, the rolling circle phenomenon is avoided, the gait is normal, and the mouse with symmetrical bilateral reaction after palpation is scored as 0; the body is slightly asymmetric, the body moves towards one side obliquely, the limbs on one side are stiff and inflexible, and the mouse with slightly asymmetric after palpation is scored as 1; moderate asymmetry, occasional circling to one side, lameness of one limb, significantly asymmetric mice scored 2 after palpation; the body is obviously asymmetric, the body turns to one side all the time, the limbs shake and slide, the limb reaction on the same side disappears after the palpation, and the mouse with weakened lateral reaction is scored as 3; the body is extremely asymmetric, the mouse rotates around a shaft, swings or does not move, the mouse is static and does not walk, and the mouse with slow sensation of limbs on both sides after palpation is scored as 4;

for step (2): normal climbing mice scored 0; crawling labored, mice with weak limbs scored 1; a mouse which is stationary on an inclined plane and cannot move up and down is scored as 2; mice that slide down the incline slowly scored 3; mice that slide rapidly without resistance scored 4;

for step (3): mice that appeared normal scored 0; slightly asymmetric mice scored 1; a significantly asymmetric mouse score of 2; a significantly asymmetric mouse score of 3; mice that were significantly asymmetric and had no mobility in their forelimbs or limbs scored 4;

for step (4): mice that appeared normal scored 0; mice that tended to turn to one side scored 1; mice circling to one side scored 2; mice turned to one side slowly scored 3; mice with immotile limbs scored 4;

the neurological deficit score totals 28 points, and the score of more than 0 is the mouse with successful model building.

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a modeling method of a mouse hypertensive cerebral hemorrhage model.

Background

The intraparenchymal hemorrhage is the second most common type of stroke, has the characteristics of high morbidity, high mortality and high disability rate, and accounts for about 20-30% of all strokes in China. Hypertensive cerebral hemorrhage (ICH) often occurs in the putamen and the endocyst regions of the basal nucleus, accounting for about 70% of ICH, with putamen bleeding often invading the endocyst, often with focal contralateral hemiplegia, hemiparesthesia and homeostatic hemianopsia. Cerebral edema, blood brain barrier integrity damage, acrosomatic dyskinesia, and neurological deficit can result after cerebral hemorrhage. Therefore, the study on the disease outcome and the treatment scheme after cerebral hemorrhage is of great significance, and the establishment of an effective and stable animal model is the first step of the following study.

At present, two methods for cerebral hemorrhage molding are reported in the literature: and establishing a cerebral hemorrhage model by collagenase induced cerebral hemorrhage model and intracranial autologous blood injection. The collagenase induced cerebral hemorrhage model is established by injecting collagenase into the right basal zone of a mouse through a stereotaxic instrument. Bacterial collagenase is a protease that dissolves and weakens the extracellular matrix surrounding the capillaries of the brain, leading to rupture of the blood vessels and subsequent extravasation of blood. However, hematoma volume is not well controlled, and diffuse bleeding is generated, which is not beneficial to experimental contrast research. And the higher mortality rate limits the application of this model. In addition, collagenase has neurotoxicity, can amplify inflammatory reaction, influences the establishment of a model, and cannot well simulate pathological changes and disease outcome of human cerebral hemorrhage. In another intracranial autoblood injection cerebral hemorrhage model, after the autoblood is extracted from a mouse, the autoblood is injected into a basal ganglia region by using a cerebral positioning instrument, and the literature reports that blood is extracted from a rat tail vein and periorbital, but the extracted blood is venous blood, and the hypertensive cerebral hemorrhage forms hematoma which is arterial blood, so the method cannot well simulate clinical hypertensive cerebral hemorrhage. Blood is also collected by a tail-cutting method, and the tail end of a rat is cut to collect blood. In addition, the method is suitable for rats, the tail artery of a mouse is positioned in the deep part of the ventral surface, is very thin and is difficult to puncture, and even if the puncture is successful, the blood taking amount is limited, so that the experimental requirement cannot be well met.

In summary, the existing molding technology has the following disadvantages and shortcomings: (1) it is impossible to find the optimal puncture site and puncture channel in the mouse, and a sufficient amount of arterial blood is drawn from the mouse for the experiment. (2) The survival rate of the mice cannot be guaranteed after puncture and blood sampling. (3) The method can not form stable hematoma, thereby providing no guarantee for subsequent experiments, being not beneficial to the contrast research of experiments and not well simulating the pathological changes and disease outcome of human cerebral hemorrhage. Therefore, the method has important significance for exploring a new optimized cerebral hemorrhage modeling method.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the modeling method of the mouse hypertensive cerebral hemorrhage model, which can not reduce the survival rate of the mouse on the premise of extracting enough arterial blood, can form stable hematoma, provides guarantee for subsequent experiments and is beneficial to the contrast research of the experiments.

The invention is realized by the following technical scheme:

a molding method of a mouse hypertensive cerebral hemorrhage model comprises the following steps:

step 1) left ventricle puncture blood sampling: the mouse is anesthetized by isoflurane gas induction, the mouse is placed in a supine position, and four limbs of the mouse are fixed, so that the chest and the upper abdomen of the mouse are fully exposed; sterilizing the chest and the upper abdomen of the mouse in a large range, and then puncturing and drawing blood at the included angle between the xiphoid process of the sternum and the costal bone by inclining 10-20 degrees to the apex of the heart to rapidly transfer arterial blood for later use;

step 2) exposure of the cranioplasty field: making a 1cm long midline incision on the scalp of a mouse with a scalpel, wiping and cleaning the soft tissue covering the skull to expose the vertical intersection point, i.e. the coronal and sagittal suture intersection point;

step 3), determining a skull surface needle insertion point: mounting the microsyringe on an injection pump, and stereoscopically orienting a needle head to a coronal and sagittal suture intersection point and zeroing; next, the stereotactic manipulator arm was adjusted to place the needle 0.2mm in front of the intersection point of the coronal and sagittal sutures and 2mm lateral to the right; at this coordinate a variable speed drill was used to drill a hole in the skull of the mouse;

step 4), injecting autoblood by using a brain stereotaxic apparatus: rapidly extracting the arterial blood reserved in the step 1), transferring the arterial blood into a glass cylinder of a microsyringe, pushing a needle head to the ventral side by 3.7mm through a drill hole on the surface of the skull of the mouse, and injecting autologous arterial blood to the caudal putamen of the right side;

step 5) standing the microsyringe: after injection is finished, the needle head is left in the original position for 10min, then the needle head is drawn back at the speed of 1mm/min, blood returning of the puncture channel is reduced, and the drilled hole is sealed by bone wax;

and 6) disinfecting the incision and suturing the skin.

Preferably, the mouse is a KM mouse, derived from swiss mice.

Preferably, the injection speed of the autologous arterial blood injected in the step 4) is 2 μ L/min.

Preferably, when the skin is sutured in the step 6), the sutured part is taken care of preventing infection, and normal saline solution is properly injected into the abdominal cavity of the mouse for supplementing liquid.

Preferably, step 7) is further included: and (3) evaluating the mice by adopting an additive removal test or scoring the neurological deficit 24h after operation, wherein the mice meeting the evaluation/scoring standard are the mice successfully modeled.

Preferably, the step of the additive removal test is as follows: attaching 0.3cm × 0.4cm of adhesive tape to the injured forepaw on the opposite side of the mouse; measuring the tactile motion response by recording the time for removing the adhesive tape, wherein the maximum observation time is 120 s; mice successfully molded are obtained when the time for removing the adhesive tape is more than 10 s.

Preferably, the step of scoring neurological deficit is as follows:

(1) placing the mouse on the surface of an empty platform to freely move for 2-5 min;

(2) placing the mouse on a 45-degree grid inclined plane for 60 s;

(3) lifting the tail of the mouse and standing upside down for 30 s;

(4) lifting the tail of the mouse and standing upside down, and placing the forelimb of the mouse on a table top for 30 s;

the scoring criteria for the neurological deficit score are as follows:

for step (1): the body symmetry is normal, the rolling circle phenomenon is avoided, the gait is normal, and the mouse with symmetrical bilateral reaction after palpation is scored as 0; the body is slightly asymmetric, the body moves towards one side obliquely, the limbs on one side are stiff and inflexible, and the mouse with slightly asymmetric after palpation is scored as 1; moderate asymmetry, occasional circling to one side, lameness of one limb, significantly asymmetric mice scored 2 after palpation; the body is obviously asymmetric, the body turns to one side all the time, the limbs shake and slide, the limb reaction on the same side disappears after the palpation, and the mouse with weakened lateral reaction is scored as 3; the body is extremely asymmetric, the mouse rotates around a shaft, swings or does not move, the mouse is static and does not walk, and the mouse with slow sensation of limbs on both sides after palpation is scored as 4;

for step (2): normal climbing mice scored 0; crawling labored, mice with weak limbs scored 1; a mouse which is stationary on an inclined plane and cannot move up and down is scored as 2; mice that slide down the incline slowly scored 3; mice that slide rapidly without resistance scored 4;

for step (3): mice that appeared normal scored 0; slightly asymmetric mice scored 1; a significantly asymmetric mouse score of 2; a significantly asymmetric mouse score of 3; mice that were significantly asymmetric and had no mobility in their forelimbs or limbs scored 4;

for step (4): mice that appeared normal scored 0; mice that tended to turn to one side scored 1; mice circling to one side scored 2; mice turned to one side slowly scored 3; mice with immotile limbs scored 4;

the neurological deficit score totals 28 points, and the score of more than 0 is the mouse with successful model building.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention adopts left ventricle puncture to take arterial blood, the puncture method can take enough arterial blood to meet the experimental requirement, and the survival rate of the mouse after blood taking can not be reduced.

2. The mouse brain locator is used for accurately injecting the autologous arterial blood back into the caudal putamen of the basal ganglia region in the brain to form stable hematoma, so that the method provides guarantee for subsequent experiments, is beneficial to experimental contrast research, and is very similar to pathological changes and disease outcome of clinical hypertensive patients, so that the method is a good molding method for the hypertensive cerebral hemorrhage model.

Drawings

FIG. 1 is the coronal section of the dissected brains of the mice from Sham group and ICH group on days 1, 2, and 3 in example 2: (1) the day of the Sham group, (2) the day of the ICH group, (3) the day of the ICH group, (2) the day of the ICH group, and (4) the day of the ICH group;

FIG. 2 is a statistical chart of the cerebral edema coefficients of the mice of the Sham group and the ICH group in example 3: (^****P＜0.0001，n＝5)；

FIG. 3 shows the results of the permeability of Evans blue between the Sham group and the ICH group in example 4: (^**P＜0.05，n＝5)；

FIG. 4 shows the additive removal test results of the mice of Sham group and ICH group in example 5 (^*P＜0.05，n＝5)；

FIG. 5 is a statistical chart of Neurological deficit score of the mice of the Sham and ICH groups in example 6: (^****P＜0.0001，n＝5)。

Detailed Description

The invention is described in further detail below with reference to specific embodiments and with reference to the following drawings.

Example 1

A molding method of a mouse hypertensive cerebral hemorrhage model comprises the following specific steps:

(1) left ventricular puncture blood sampling: the mouse is anesthetized by isoflurane gas induction, the mouse is placed on a hard board in a supine position, four limbs of the mouse are fixed by medical adhesive tapes, cotton threads are buckled on the front teeth of the mouse, and the mouse is pulled and fixed, so that the chest and the upper abdomen of the mouse are fully exposed. Dipping 75% standard medical alcohol by using a cotton swab, disinfecting the chest and the upper abdomen in a large range, touching the heart most intense point (apex of the heart) of the chest with the left hand, puncturing the chest at the heart most intense point by using a 1mL syringe at the included angle of the xiphoid process of the sternum and the costal bone and inclining by 10-20 degrees to aim at the heart most intense point, withdrawing the needle and withdrawing blood, and quickly transferring arterial blood to a 1.5mL EP tube for later use.

(2) Exposing a craniotomy field: a1 cm long midline incision was made on the scalp with a scalpel, and the soft tissue covering the skull was cleaned with a cotton swab dipped in hydrogen peroxide to expose the vertical intersection, the coronal and sagittal suture intersection (bregma).

(3) Determining the needle insertion point on the surface of the skull: a microsyringe (50 μ L) was mounted to the syringe pump and the needle was stereotactically oriented to bregma and zeroed. Next, the stereotactic manipulator arm was adjusted to place the needle 0.2mm before the bregma point and 2mm to the right side. At this coordinate a variable speed drill with a 0.6mm drill bit was used to drill a hole in the skull.

(4) Injecting autoblood by a brain stereotaxic instrument: 20 mul of arterial blood (the volume of the blood can be adjusted according to the molding requirement) is rapidly extracted from an EP tube and transferred into a glass cylinder of a microsyringe, a needle head passes through a drill hole on the surface of the skull and is pushed 3.7mm towards the ventral side, namely the position of the caudal putamen on the right side can be reached, and 20 mul of autologous arterial blood is injected at the speed of 2 mul/min.

(5) Standing the microsyringe: after the injection is completed, the needle is left in place for 10min and then withdrawn at a rate of 1mm/min to reduce the blood return from the puncture, and the drilled hole is sealed with bone wax.

(6) The incision is sterilized and the skin is sutured.

And (3) postoperative treatment: the suture is taken care of preventing infection, and normal saline is properly injected into the abdominal cavity for fluid infusion.

Example 2 post-modeling hematoma coronary surface of hypertensive cerebral hemorrhage in mice

10 male Kunming mice (derived from swiss mice) with the age of 5-7 weeks are divided into two groups (n is 5) with the weight of 20-30 g, and the two groups are respectively a Sham group and an ICH group, and are raised in a quiet environment, and are taken with free water. In the Sham group, a microsyringe was inserted only 3.7mm from the drilled hole to the ventral side without autologous blood extraction, the needle was left in place for 10min before needle withdrawal, then withdrawn at a rate of 1mm/min, the drilled hole was sealed with bone wax, and the skin was sutured. The ICH group is executed according to the specific steps of the molding method described in embodiment 1. After the model is made, the coronal plane of the Sham group and the coronal plane of the ICH group hematoma are observed, the ICH group coronal plane is coronal-incised by taking the injection point as the central point, 1, 2 and 3 days are selected as the observation time points, and the coronal plane after the coronal-incising is shown in figure 1.

As shown in FIG. 1, the coronal section of the Sham group (FIG. 1(a)) showed only microsyringe puncture, the coronal section of the ICH group showed a clear, stable hematoma after molding, with the volume of the hematoma being maximal on day 1 (FIG. 1(b)) and then decreasing over time (FIG. 1(c) -FIG. 1 (d)). These changes are consistent with pathological changes and disease outcome that hematoma in basal ganglia region is gradually absorbed and dissipated after clinical hypertensive cerebral hemorrhage.

Example 3 measurement of the degree of cerebral edema after modeling of hypertensive cerebral hemorrhage in mice

10 male Kunming mice with the age of 5-7 weeks are divided into two groups (n is 5) with the weight of 20-30 g, wherein the two groups are respectively Sham and ICH, and are raised in a quiet environment and freely drunk by drinking water. In the Sham group, a microsyringe was inserted only 3.7mm from the drilled hole to the ventral side without autologous blood extraction, the needle was left in place for 10min before needle withdrawal, then withdrawn at a rate of 1mm/min, the drilled hole was sealed with bone wax, and the skin was sutured. The ICH group is executed according to the specific steps of the molding method described in embodiment 1. Then, carrying out intraperitoneal injection of 10% chloral hydrate for inducing anesthesia on the mice of each group for 24h by adopting a dry-wet weight method, cutting off heads, taking out brains, removing olfactory bulbs, cerebellums and brainstems, immediately weighing to obtain wet weights, then drying the brain tissues in an oven at 60 ℃ for 72h, and weighing the brain tissues to obtain dry weights after the brain tissues are restored to room temperature. The brain water content (%) of each group of mice was calculated as (wet weight of brain tissue-dry weight of brain tissue)/wet weight × 100%. The statistical map of the measured brain edema coefficients is shown in fig. 2.

As shown in fig. 2, the brain edema coefficient of the Sham group loiters at 0.785, whereas the brain edema coefficient of the ICH group is significantly higher than that of the Sham group, which is compared with the Sham group,^****p is less than 0.0001, n is 5, and the statistical difference is obvious. Indicating that the cerebral edema degree is obviously increased after the model is made. The clinical characteristics of the cerebral edema of the patient after the clinical hypertensive cerebral hemorrhage are met.

Example 4 measurement of blood brain Barrier integrity after modeling of hypertensive cerebral hemorrhage in mice

10 male Kunming mice with the age of 5-7 weeks are divided into two groups (n is 5) with the weight of 20-30 g, wherein the two groups are respectively Sham and ICH, and are raised in a quiet environment and freely drunk by drinking water. In the Sham group, a microsyringe was inserted only 3.7mm from the drilled hole to the ventral side without autologous blood extraction, the needle was left in place for 10min before needle withdrawal, then withdrawn at a rate of 1mm/min, the drilled hole was sealed with bone wax, and the skin was sutured. The ICH group is executed according to the specific steps of the molding method described in embodiment 1. Then, 2% Evans blue was prepared with physiological saline. The injection amount of the mouse is 4mL/kg after 24 hours of cerebral hemorrhage, 2% Evans blue is injected through tail vein, and the perfusion is carried out for 3 hours. Bleeding lateral brain tissue weighing: the 10% chloral hydrate is used for inducing anesthesia after intraperitoneal injection, after a mouse is completely anesthetized, the chest is opened, the heart is perfused by 50mL of physiological saline, the brain tissue is completely taken out from the skull by cutting off the head, the olfactory bulb, the cerebellum and the brainstem are removed, the cerebral hemisphere on the blood side is taken out, and the wet weight of the brain tissue is weighed.

And (3) extraction: placing the cerebral hemisphere at the bleeding side into an EP tube filled with 3mL of precooled formamide, sealing the EP tube by using a plastic package membrane, placing the sealed EP tube in a water bath kettle at 60 ℃ for incubation for 72 hours, centrifuging the sample at the rotating speed of 3000r/min for 10min, and taking out the supernatant for later use.

And (3) determining the content of the Evans blue:

(1) standard product of evan blue: mu.g of Evans blue was taken, a volume of 1mL was determined with formamide, and Evans blue was diluted to a concentration of 20. mu.g/mL.

(2) The standard was added to the standard wells of a 96-well plate at 200. mu.L, 100. mu.L, 50. mu.L, 25. mu.L, 12.5. mu.L, 6.25. mu.L, 0. mu.L, and a formamide replenishment volume of 200. mu.L was added to the standard wells. To the sample wells of the 96-well plate, 200. mu.L of the sample was added as shown in Table 1 below.

TABLE 1 Evans blue concentration determination of sample loading

(3) And (3) measuring absorbance: and detecting OD values of each group of standard products and samples at 632nm wavelength by using an enzyme-labeling instrument, drawing an EB concentration standard curve, calculating the content of the evans blue of the samples to be detected according to the OD values of each group of samples, dividing the content by the wet weight of the bleeding side brain tissue, and measuring the permeability of each group of evans blue so as to prompt the permeability of the blood brain barrier. The results of the measured evans blue permeability are shown in fig. 3.

Results as shown in fig. 3, the evans blue permeability values for the Sham group loitering at 1.349, while the evans blue permeability values for the ICH group loitering at 9.415 were significantly higher than those for the Sham group, which was compared to the Sham group,^**p is less than 0.05, n is 5, and the difference has statistical significance. The higher the permeability of the evans blue after the model is made, the higher the permeability of the blood-brain barrier, namely the integrity of the blood-brain barrier is destroyed. The blood brain barrier integrity of the patient can be damaged after the hypertensive cerebral hemorrhage is clinically met.

Example 5 measurement of tactile and motor functions of mice after modeling of hypertensive cerebral hemorrhage

10 male Kunming mice with the age of 5-7 weeks are divided into two groups (n is 5) with the weight of 20-30 g, wherein the two groups are respectively Sham and ICH, and are raised in a quiet environment and freely drunk by drinking water. In the Sham group, a microsyringe was inserted only 3.7mm from the drilled hole to the ventral side without autologous blood extraction, the needle was left in place for 10min before needle withdrawal, then withdrawn at a rate of 1mm/min, the drilled hole was sealed with bone wax, and the skin was sutured. The ICH group is executed according to the specific steps of the molding method described in embodiment 1. The additive removal test was used to evaluate the haptic response and sensorimotor performance 24h after molding. 0.3cm by 0.4cm of tape was applied to the contralateral injured forepaw. The tactile motor response was measured by recording the time to remove the tape, with a maximum observation time of 120s, and a time to remove the tape > 10s for successfully molded mice. The obtained additive remove test results are shown in FIG. 4.

Results as shown in fig. 4, the additive remove test time of Sham group loitering at 6.8s while the test time of ICH group loitering at 74s was significantly higher than Sham group, which was compared to Sham group,^*p is less than 0.05, n is 5, and the difference has statistical significance. Indicating that the tactile and motor functions are weakened after molding. The physical examination characteristics of the limb touch sense and the motor function weakening of the patient after the clinical hypertensive cerebral hemorrhage are met.

Example 6 mouse neurological deficit score after hypertensive cerebral hemorrhage modeling

10 male Kunming mice with the age of 5-7 weeks are divided into two groups (n is 5) with the weight of 20-30 g, wherein the two groups are respectively Sham and ICH, and are raised in a quiet environment and freely drunk by drinking water. In the Sham group, a microsyringe was inserted only 3.7mm from the drilled hole to the ventral side without autologous blood extraction, the needle was left in place for 10min before needle withdrawal, then withdrawn at a rate of 1mm/min, the drilled hole was sealed with bone wax, and the skin was sutured. The ICH group is executed according to the specific steps of the molding method described in embodiment 1. Mice were scored for Neurological deficit at 24h postoperatively according to the 28-point Neurological deficit scoring scale (Neurological deficit score), as shown in table 2 below, with a score > 0 for successfully molded mice.

TABLE 2 mouse 28 points neurological impairment score Scale

The statistical results of the obtained Neurological deficit score are shown in FIG. 5.

Results as shown in fig. 5, Neurological deficit score of Sham group was 0, while score of ICH group lingered significantly higher at 12.4 than Sham group, which was compared to Sham group,^****p is less than 0.0001, n is 5, and the statistical difference is obvious. Indicating that the nerve function defect is serious after the model is made. The method is in line with the neurological score and physical examination characteristics of neurological impairment of patients after clinical hypertensive cerebral hemorrhage.